Locating marker/tracer elements detectable by neutron activated analysis within or on carrier microspheres, including microspheres used in biological experimentation

ABSTRACT

Microspheres are permanently marked with non-radioactive stable isotopes of elements suitably detected by neutron activation analysis. The marked microspheres are suitable to permanently label diverse things. For example, families of stable-multiple-isotope-marked microspheres injected into an animal become lodged by the circulating blood within selected tissues of an animal during blood flow analysis experimentation. Absolute and relative abundances of these stable-isotope-marked microspheres residing within harvested tissues are readily accurately automatically measured in situ within the harvested tissue samples by neutron activation analysis. The quantitatively measured abundance of the isotopes, and associated microspheres, are accurately indicative of the former flow of blood containing the microspheres to the tissue. Microspheres are preferably marked with stable isotopes of gold, antimony, lanthanum, samarium, europium, terbium, holmium, ytterbium, lutetium, hafnium, tantalum, tungsten, rhenium, osmium, iridium, scandium and/or bromide.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention generally pertains to chemical or elementalmarkers or tracers that, when combined with other chemical admixtures orcompounds, or when inserted into or upon objects or devices, thereafterserve to permanently identify such admixtures, compounds, objects ordevices, including after such change(s) and gross change(s) to thecompounds, objects or devices in form and/or in composition as may beoccasioned by lapse of time, dissipation, wear, deterioration,oxidation, or explosion.

[0003] The present invention particularly concerns (i) elemental orchemical markers that are indefinitely long lasting, and detectable atthe level of a single atom or molecule over a fast range of densities;(ii) the use of neutron activation in the detection of elemental andchemical markers, and the elemental or chemical markers so detectable;and (iii) the packaging of, and/or carriers for,neutron-activation-detectable elemental and/or chemical markers,including the packaging of elemental and/or chemical markers in carriermicrospheres, including microspheres as are used in biologicalexperimentation including, inter alia, in blood flow analysis.

[0004] 2. Description of the Prior Art

[0005] 2.1 Neutron Activation Analysis

[0006] The present invention will be seen to employ neutron activationanalysis.

[0007] Individual stable elements (e.g., gold) are known to haveisotopes that are strongly detectable by neutron activation analysis.The known abundance of the “marker” elements in certain substances haspermitted these elements to serve as markers for the substances. SeeKennelly, J. J., Apps, M. J., Turner, B. V. and Aherne, F. X. 1980;Dysprosium, cerium and chromium marker determination by instrumentalneutron activation analysis. Can. J. Anim. Sci. 60:749-761. See alsoNishiguchi Y., Sutoh M., Nishida T., Satoh H., Miyamoto S.; Neutronactivation analysis of Lanthanides (La, Sm and Yb) as a particle marker,and estimation of passage rates. Anim. Sci. Technol. (Jpn.), 67:787-793. 1996.

[0008] Neutron activation analysis can provide the life sciencecommunity with capabilities not readily available with other assaytechnologies. Neutron activation is well known for both (i) itsexcellent sensitivity and (ii) its specificity for the simultaneousmeasurement of multiple elemental trace elements. This specificityoffers the potential of being able to measure multiple isotopic tracersper assay.

[0009] Unlike light, neutrons can penetrate solid tissue andopaque-liquid samples, thereby providing an assay that is completelyself-contained with but minimal sample preparation.

[0010] Unlike other element detection methods, such as atomic absorptionspectrophotometry, neutron activation is not chemically or physicallydestructive. Therefore, samples can be archived, re-assayed, and/orundergo additional chemical analysis following neutron activation.

[0011] Because only the samples of interest are neutron activated, assayby neutron activation analysis can significantly reduce occupationalexposure to radiation and eliminate the low-level radioactive wastegenerated. For example, the contamination of gloves and protectiveclothing, glass and plastic laboratory supplies, and waste products fromresearch animal housing and carcasses attendant upon the use ofradioactive tracers are completely avoided. Stable isotopes do notundergo radioactive decay or cause radiokinistics and, unlike somecolorometric probes, they do not suffer any loss of activity (i.e., lossof fluorescence) over time. Therefore, stable isotope labeled productswill have an indefinitely long shelf life: significantly longer thancompeting labeling methods.

[0012] The major disadvantage to assay by neutron activation technologyis the required access to a neutron source. If the neutron radiationsource is to be strong (i.e., with a high flux) so that it will excite asignificant proportion of the target tracers—preferably stableisotopes—of the sample—which sample may be sparse—to an excited,radioactive, energy level in a reasonable time, providing thereby areasonable population of radionuclides the decay of which may likewisebe detected during a reasonable time, then the source of neutron fluxmust most commonly be energetic, as is typically derived from a researchreactor. Suitable research reactors, and reactor time, are not scarce inthe United States circa 1999. However, the reactors are located atparticular sites not normally coincident with sites at whichinvestigations in the life sciences are conducted. Therefore, samplesfor assay by neutron activation analysis must normally be sent to areactor, irradiated with neutron flux, and analyzed with results beingreported to the sender. Furthermore, the samples, if not permanentlyarchived at or near the site of the reactor, may be returned to thesender only when radiation has sufficiently abated.

[0013] Accordingly, and despite the many advantages of neutronactivation, this general analytical tool has not (as of 1999) reachedits full potential within the life science community due to somecombination of (i) a lack of user awareness of the technique, (ii) alack or perceived lack of access to reactors, and/or, importantly to thepresent invention, (iii) a lack of commercially available stable-labeledresearch products specifically designed for neutron activationtechnology. These research products would desirably be targeted onintended biological research applications, and be of a form familiar tobiological researchers.

[0014] Most recently, Biophysics Assay Laboratory, Inc., 280 WellesleyAvenue, Wellesley Hills, Mass. 02481 (Phone/Fax: (781) 239-0501)[“BioPAL”] has been formed to (i) develop, manufacture and market a newgeneration of high-precision stable-labeled research products, and to(ii) provide a state-of-the-art assay service that can meet commercialdemand. The present invention will be seen to concern thesestable-labeled research products, developed jointly with TritonTechnology, Inc., of San Diego, Calif.

[0015] In passing, it should be noted that neutron activation analysisalso has an extensive history in the detection of explosives—which is adifferent thing than the detection of stable isotopes, used as makers,that may be placed into, inter alia, explosives as will be taught by thepresent invention. In other words, certain chemicals present in certainexplosives can be directly detected by neutron activation analysis. As aleading book on this topic, see Explosive detection using fast neutronactivation analysis by Terry E. Carrell, published by North AmericanRockwell, Los Angeles, Calif.

[0016] When the present invention is later understood to bestable-isotope labeled microspheres usable as markers in diversecircumstances, it will be useful to consider the possible use of thesemarkers in labeling explosives. Stable-isotope labeled microspheresserving as identifying markers cannot be assured to be emplaced inexplosives save those legitimately produced, and then only under mandateof law or regulation.

[0017] However, in accordance with the general principles of the presentinvention, later explained, to the effect that the carriage, and thechemistry, of the marker stable isotope is divorced from the chemistryof the exterior surface of a microsphere which serves to mechanicallyretain the marker stable isotope, it can be anticipated, in advance,that ubiquitous, tailored, stable-isotope labeled microspheres inaccordance with the present invention will be very useful forpermanently marking an immense number of different admixtures,compounds, objects or devices, including, inter alia, explosives.Properly tailored to a target, including an explosive, about the onlyway of expunging microspheres and any elemental markers that theycontain from a compound such as an explosive is by gross moleculardissociation such as is characteristic of, inter alia, explosion. Ofcourse, to remove the microspheres, and markers, requires destruction ofthe compound. Moreover, such explosion and attendant moleculardissociation does not truly get rid of the elemental markers, whichremain (and will remain, short of atomic transmutation) as residue.

[0018] It will next immediately be discussed that microspheres may beboth (i) mechanically and/or (ii) chemically, tailored, or “targeted”,to their intended environment of use. Most of this discussion willinvolve prior art biological uses of microspheres. Stable-isotopelabeled microspheres will later be seen to be fully as susceptible ofbeing “targeted” upon an intended environment of use as were previousmicrospheres. Moreover, and recalling the requirements of labelingexplosives, it should be appreciated in considering the diverse labelingand marking requirements of the prior art (both biological andnon-biological), that the “targeting” of stable-isotope labeledmicrospheres in accordance with the present invention on an intendedenvironment of use will prove to be selectively either broad or narrow,and semi-permanent or permanent, all in accordance with (i) applicationrequirements and (ii) the principles of chemistry.

[0019] 2.2 Types of Microspheres, Circa 1999

[0020] The mechanism of the present invention for delivering stableelements that are suitable to form isotopes detectable by neutronactivation analysis will be seem to be: microspheres.

[0021] “Microspheres” is the generic term applied to certain minute,typically homogeneous and uniformly-size-graded, particles, beads orwhatever existing in over 2000 types. The microspheres are commonly madeof latex rubber, polystyrene (PS) plastic, or other polymers,copolymers, terpolymers, and silica. They come in a variety of densitiesfrom 0.9-2.3 g/ml. They come in a sizes ranging from nanometers tomillimeters.

[0022] Microspheres are both (i) mechanically and (ii) chemicallyversatile; properties of which good utility will later be seen to bemade in the present invention.

[0023] A broad range of sizes and types or microspheres—also known asuniform latex particles—are available from commercial sources. Forexample, diverse microspheres are available from Bangs Laboratories,Inc., 979 Keystone Way, Carmel, Ind. 46032.

[0024] Microspheres are available in diameters from ˜0.020 μm (20 nm) to1000 μm (1 mm). Size uniformity is excellent: c.v.'s are typically <3%,and are often ˜1.

[0025] Colored and fluorescently-colored microspheres are available fromsaid Bangs Laboratories, Inc., from Triton Technologies, Inc., and fromMolecular Probes, Inc. in a spectrum of colors. More than 300 differentdyed microspheres are available in colors from red to beyond violet, aswell as black, white, and gray. Bright primary colors are available foreasy color identification and mixing of colors; others are very dark,like ink, for good contrast.

[0026] Microspheres may be obtained that are dyed with fluorescent dyes.More than 30 different-sized microspheres colored with >10 differentabsorbance and fluorescent dyes exhibiting various excitation andemission wavelengths are commercially available.

[0027] Microsphere surface chemistries range from hydrophobic (plainpolystyrene) to very hydrophilic surfaces imparted by a wide variety offunctional surface groups: 1) aldehyde —CHO; 2) aliphatic amine—CH2-NH2; 3) amide -CONH2; 4) aromatic amine—NH2; 5) carboxylic acid—COOH (3 different types); 6) chloromethyl —CH2—Cl; 7) epoxy; 8)hydrazide —CONH—NH2; 9) hydroxyl —OH; 10) sulfate —SO4; and 11)sulfonate -SO3.

[0028] The original recipe for various types of ˜1 μm (100 nm) diameterCOOH— or —NH2 modified microspheres contained 12, 20, 40, or 60%magnetite. Truly superparamagnetic, these microspheres respond to amagnet but display no residual magnetism. These microspheres can be usedfor direct adsorption of proteins, or surface groups can be used forcovalent coupling of ligands (proteins, DNA, etc.)

[0029] The narrow size distribution type were designed for betterperformance in cell depletion applications. Encapsulated microsphereshave a magnetite-rich core and a polystyrene shell, with COOH and NH2surface groups. These microspheres make better solid supports forapplications using enzymes because there is no iron on the surface.

[0030] As an example of microspheres with attachments, the ProActiveStreptavidin coated superparamagnetic beads of Molecular Probes, Inc.,serve as a generic magnetically responsive solid phase to which avariety of biotinylated items can be attached. The ProActive Protein Acoated magnetic microspheres provide an IgG-binding affinity supportthat is extremely easy to manipulate.

[0031] For example, ProActive GAM magnetic microspheres having goatanti-mouse Fc-specific IgG serve to bind mouse IgG's and orient themouse IgG's correctly for high activity with less primary Ab.

[0032] Streptavidin, protein A, and GAM coated non-magnetic, polymericmicrospheres are also available in several sizes. Some of the morecommon applications of protein-coated microspheres include: (i) affinitychromatography, (ii) multi-purpose solid phase for immunoassays, (iii)nucleic acid hybridization, (iv) immunoselective cell separation, and(v) DNA sequencing.

[0033] For purposes of the present invention, it should only beunderstood that microspheres can both (i) mechanically carry diverseelements within their matrix—for example, ferromagnetic iron—and can(ii) chemically affix diverse chemical compounds, including those ofbiological interest. Both the (i) mechanical and (ii) chemicalcombinations can be relatively permanent, essentially demanding adestruction of the microsphere (which may not be easy) in order to severthe association.

[0034] 2.3 Uses Of Microspheres, Circa 1999

[0035] 2.3.1 Blood Flow Analysis

[0036] Microspheres of both the radioactive and the non-radioactive,absorbance-dye and fluorescent-dye labeled, types are regularly used inthe measurement and analysis of in vivo blood flow or, moreparticularly, regional myocardial blood flow (RMBF). Such microspheresare also used in measurement of the flow of gas in the lungs, or themovement of materials through the gut, or like fluid movement processesoccurring within the higher animals.

[0037] As regards the use of radioactive-labeled microspheres, theirpresence is detected with detectors of gamma radiation emitted duringdecay events.

[0038] As regards the use of non-radioactive absorbance-dyed orfluorescently-dyed microspheres, the dyes used to mark the microspheresmay be, in different variants, either elutable to non-elutable, with thedye absorbance or fluorescence measured in various ways, including inbulk by the use of automated or semi-automated absorbance orfluorescence detector equipments.

[0039] 2.3.2 Latex Agglutination Tests and Particle Immunoassays

[0040] Many microspheres are used in various medical diagnosticapplications. Proteins will adsorb readily onto polystyrene (PS)microspheres or they may be covalently coupled to carboxylic acid orother surface functional groups. Microspheres so coated with antibodiescan be agglutinated (agglomerated) by the appropriate antigens.

[0041] 2.3.3 Sandwich Assays and Tests (Particle Capture ELISA's)

[0042] Antibody-coated microspheres form the basis for particle captureELISA tests and related assays (i.e., those that form a blue dot).Antigen links the sandwich of (1) primary antibody-coated particle and(2) enzyme-labeled secondary antibody. Microspheres permit easypreparation of reagents (antibody coating of microspheres done in bulk,not on a membrane) and precise placement of Ab-coated microsphere spotson top of the filters.

[0043] 2.3.4 Dyed Particle Sandwich or Chromatographic “StripTests”

[0044] Darkly dyed microspheres can eliminate the need for enzymes (andtheir attendant stability problems) in sandwich assays. Tests use dyedmicrospheres attached to one of the two sandwich antibodies. Small,antibody-coated microspheres move easily through the membrane inchromatographic-like assays; deeply dyed (some as dark as ink!), theybring enough color to the sandwich to completely preclude the use ofenzymes. A wide variety of colors are available, including brightfluorescents.

[0045] 2.3.5 NIST-traceable Standards

[0046] Calibrated uniform diameter microspheres for use as standards andcontrols for particle or cell counting and measuring instruments areavailable singly or in sets.

[0047] 2.3.6 LDV/PIV Seeds

[0048] Microspheres have been used as seed in fluid flow streams forlaser Doppler and particle image (and other) velocimetry measurements.In these applications they maybe dispersed in gas streams, wind tunnels,wave tanks, or ship tow-tanks. Silica microspheres permit use incombustion studies and other high temperature applications, too.

[0049] 2.3.7 Other Applications

[0050] Other applications for microspheres include 1) blood cellsimulation; 2) cell separation; 3) phagocytosis studies; 4)chemiluminescent assays; 5) column packing (non-porous) ; 6) densitycalibrators; 7) DNA probes/ PCR; 8) instrument standards; 9) fluidizedbeds; 10) magnetic resonance imaging; 11) model studies; 12) solid phaseDNA sequencing; 13) spacers for flat panel displays; and 14) voidsources for ceramics.

[0051] Applications for dyed microspheres include 1) stains; 2)adjuvants; 3) contrast agents; 4) cell tags (rosettes) ; 5) gelpermeation markers; 6) flow markers for liquids; and 7) confocalmicroscopy standards.

[0052] 2.4 Methods of Using and Measuring Microspheres, Circa 1999

[0053] General background to the present invention as regards the use ofmicrospheres in biological measurements may be found in U.S. Pat. No.5,230,343 for COLORED MICROSPHERES FOR MEASURING AND TRACING FLUIDMIXING AND FLOW, PARTICULARLY BLOOD FLOW TO TISSUE to inventors GerdHeusch, Michael P. Guberek, and W. Scott Kemper; in U.S. Pat. No.5,253,649 for a PROCESS FOR THE MEASUREMENT OF BLOOD CIRCULATION BYMEANS OF NON-RADIOACTIVE MICROSPHERES to inventors Gerd Heusch, RainerGross, Wolfgang Paffhausen and Andreas Schade; and in German patentapplication Serial No. P 40 19 025.0 filed in Germany on Jun. 14, 1990which is the priority application to both these patents. All theserelated patents are assigned to Triton Technology, Inc., of San Diego,Calif., a corporation of the State of California. The contents of therelated patents are incorporated herein by reference.

[0054] 2.4.1 Use of Microspheres in Fluid Flow Analysis

[0055] Traditionally, most measurements of fluid mixing and fluid(s)flow(s) are direct. One or more fluid flows may simply be measured whilesuch flows are occurring. Alternatively, any mixture that results fromthe flows of two or more fluids may be analyzed as to its constituentcomponents in order to quantitatively determine the fluid flows thathave transpired.

[0056] However, direct measurement of fluid(s) flow(s), such as withinthe blood stream of a living animal, is often impossible. Moreover,direct quantitative analysis of the constituent components of complex,or extensive, mixtures of fluids is often prohibitively difficult orexpensive. The expense is magnified if many samples must be taken, andanalyzed, over time.

[0057] Accordingly, modeling or simulation is sometimes used in order totrace the flow, and mixing, of one or more fluids. For example, a dyemay be put in ground water and its dispersion may subsequently beobserved. From the observed dispersion of the dye a similar dispersionof pollutants, or other less readily detectable fluids, may be imputed.

[0058] Another, relatively sophisticated, form of fluid flow and fluidmixing analysis is indirect. A physical marker is put into, or achemical marker is bonded to, an actual fluid, or a fluid component, forwhich flow and/or mixing is desired to be assessed. The fluid serves asa carrier. When the distribution of the marker is analyzed then thecorresponding distribution of the carrier fluid is imputed.

[0059] The highest, and most exacting, expression of this indirectmethod is in medicine, and particularly in blood flow analysis. Theblood, and the organs and tissues receiving blood, within a livinganimal present a system that is very complex in its fluid flow patternsand dynamics, and that is difficult of direct access and measurement.Accordingly, microscopic markers are placed by catheter into the leftatrium of the animal's heart, entering into the animal's blood throughlymixed where they are subsequently distributed to the animal's tissues inproportion to the blood flowing to the tissues.

[0060] The microscopic markers are commonly microspheres sized(typically less than 30 μm and more than 7 μm and more typically 15 μm)so that they are trapped by, and permanently lodge within, the smallestcapillaries of the animal's tissues. In proportion to the flow of blood,the particles are sized so that they are trapped in the capillaries ontheir first pass through the circulation system of the animal. Thetissues are subsequently harvested, and the prevalence—i.e., the numbers—of the markers have previously been analyzed, producing thereby anindirect indication of the blood flow to the tissue.

[0061] Previous systems developed for medical blood flowanalysis—discussed in greater detail hereafter—have proven to be bothcomplex and expensive. Because of their cost and complexity, suchsystems have not been found suitable for use in routine industrial orenvironmental fluid flow and mixing measurement problems.

[0062] However, it should be recognized that the flow of blood, or bloodcomponents, within the arteries and veins of a living animal is onlydifferent in complexity, and not in the essential nature of fluid flowdynamics, from the flows of fluids occurring within factories,ecosystems, and the like. Accordingly, if a reliable, effective,inexpensive, and automated (or semi-automated) indirect fluid flowmeasurement system suitable for use on the difficult problem of bloodflow analysis could be developed, then such a system might well havegeneral applicability to the tracing and measurement of fluid flows, andthe mixing of fluids, in many other diverse applications.

[0063] 2.4.2 The Earliest Measurements of Blood Flow With RadioactiveMicrospheres

[0064] The reasons for the measurement of blood flow are set forth inU.S. Pat. No. 4,616,658 to Shell, et. al., for NON-RADIOACTIVELY LABELEDMICROSPHERES AND USE OF SAME TO MEASURE BLOOD FLOW. Shell and hisco-inventor See teach a safe and inexpensive method of measuring bloodflow in experimental animals using non-radioactively labeledmicrospheres is provided. The microspheres may be comprised of a varietyof materials, including latex and agarose, and may be labeled withcolored dyes or by linkage to enzymes, plant enzymes being preferredbecause they do not occur naturally in an animal's system. Afterinjection and circulation of the microspheres throughout the animal'ssystem, blood flow to particular tissue may be measured by counting thenumber of microspheres in the tissue sample, the initial number ofmicrospheres in the animal's blood stream having been measured shortlyafter injection. In the case of microspheres labeled with colored dyes,the spheres may be counted in tissue either after separation from thetissue or while still trapped in the tissue's capillaries. Techniquesfor separating the microspheres from blood and tissue are also provided.

[0065] The measurement of blood flow in experimental animals is oftennecessary in the fields of pharmacology, physiology, therapeutics anddiagnostics. For example, toxicology studies require blood flowmeasurement to determine the toxicity of various suspected toxic agents.Further, many diagnostic and therapeutic advances have some impact onthe flow of blood. It is therefore desirable to take blood flowmeasurements.

[0066] Blood flow measurements can be performed in many anatomicalareas, including the brain, heart, lung, gut, kidney, reproductiveorgans, skin and muscle. One sensitive and specific previous techniqueinvolves the use of radioactively labeled microspheres. In one variantof the technique plastic or polystyrene microspheres are marked with aradioactive label and injected into the left atrium of the heart of anexperimental animal. They are injected into the left atrium in order toachieve homogeneous mixing of the spheres in the blood. The prevalenceof the radioactively-labeled microspheres in the blood is assessed bywithdrawal of a blood sample from the aorta downstream from the heart,during the complete course of the injection. This “reference withdrawal”sample is used to determine the “radioactivity per volume flow rate” ofthe blood coming from the heart. The mixed microspheres disperse inproportion to blood flow and lodge in the micro-capillaries within thetissues of the animal. The animal is later sacrificed and the organ(s)of interest is (are) harvested. Blood flow to a particular organ isdetermined by measuring the level of radioactivity in the organ sample,which radioactivity is a function of the number of microspheres trappedin each portion of the organ. This radioactivity level is divided by thereference withdrawal value in order to determine absolute blood flow.

[0067] Notably, the radioactive strength, or intensity, of the injectedmicrospheres is not required to be exactly known. Ultimately only ratiosbetween the (i) density of injected microspheres, and (ii) the densityof microspheres recovered from each tissue, will prove relevant. Tostart, blood is withdrawn at a predetermined rate from a site downstreamfrom the point of injection for a longer time than it takes for all theinjected microspheres to pass this point. Only a small fraction of theflowing blood, and a commensurately small fraction of the microspherescontained within the blood, are extracted. However, the density of themicrospheres within the blood is directly determinable in terms of unitsradioactivity (i.e., radioactive intensity) per unit measure of bloodflow rate (volume per unit time). Notably, it is not necessary tocalculate the numbers of microspheres per unit blood—although thisnumber may also be determined.

[0068] Later, when the animal's tissue samples are harvested, eachtissue sample obviously contains but a minute fraction of the millionsof injected microspheres that are now lodged within, and blocking, of acorresponding number of minute capillaries of the billions of suchcapillaries within the animal's entire body. The intensity ofmicrospheres within the harvested tissue may likewise be expressed interms of units radioactivity (i.e., radioactive intensity) per unitmeasure of volume or of weight. Dividing the harvested radioactiveintensity by the injected intensity causes the specific radioactivestrength of the microspheres to cancel out of the equation, and thevolume blood flow (normally expressed in ml/min/gm) reaching the organof interest is directly determined. Prior art dye-elution microspheres,which will be later discussed, work on the same principle.

[0069] Dye-colored microspheres are better adapted to long termquantification than are radioactive microspheres once a microsphere is(quantitatively) color-dyed as it then holds the dye, withoutappreciable change, during all conditions of storage and passage throughthe bloodstream.

[0070] 2.4.3 Limitations of Conventional Radioactive Microspheres

[0071] The main (but not the only) problem with radioisotope-labeledmicrospheres is shelf life. In order that the decay events from theradioisotopes should be detectable during the lapse of a reasonableperiod of time, the radioisotopes must have short half-lives. Aradioisotope lodged within a microsphere commonly has a radioactivity“half-life” that is as short as several days and no longer than a fewweeks or months; the intensity of the radioactive emission from theradioactive microsphere decreasing by half with each passing of the“half life” period. Since radioactivity decays with time, it becomesnecessary to inject larger and larger numbers of aged microspheres topermit that the microspheres should still be reliably detectable.

[0072] A supply of radioactive microspheres ages even while they are onthe shelf. Radioactive microspheres thus have a time limited shelf life,which adds a cost factor to their use. The problem of decreasingradioactive intensity does not end with injection into an animal. Caremust also be taken not to let too much time go by before harvesting andanalyzing the tissue samples or there may be insufficient activity todetermine low fluid flows due to the ‘noise’ threshold of a typicalgamma counter used for measurement of radioactivity. Constantreplenishment, inventory management, and renewal of microspheres usedin, principally, biological experimentation is an onerous laboratorytask. If not performed diligently experimental schedules may bedisrupted. Although due precautions are taken in transport and storageof radioactive microspheres, the constant flux of newly producedradioisotopes from manufacturer to laboratory, the controlled storage ofradioisotopes still suitable for experimental use, and the long term ofradioisotopes no longer suitable for use but still sufficientlyradioactive so as to be unsuitably released into the environment, allinvolve a biohazard.

[0073] The present invention will be seen to avoid this problementirely; all isotopes being distributed, stored, used, optionallystored again, and re-shipped in a non-radioactive form over anindefinitely long time period with an arbitrarily long duration in eachphase. Meanwhile, the high sensitivity and specificity of radioactivelabeling will be seen to be preserved.

[0074] Still other problems and disadvantages associated withradio-isotope labeled microspheres will be seen to be eliminated orsubstantially abated.

[0075] First, the high start-up costs of using radioactive isotopes willbe seen to be avoided. These costs commonly include special governmentlicensing, and purchase and maintenance of each of a gamma counter tomeasure radioactivity, shielding to protect laboratory workers fromradiation exposure, and complex storage facilities. There is typically ahigh minimum “per order” cost of equipments from manufacturers. Thesehigh costs severely limit the use of radioisotope-labeled microspheresin blood flow measurement, generally restricting its use to largelaboratories and medical centers.

[0076] Second, because of the half-lives of their containedradioisotopes, radioactively-labeled microspheres have a limited shelflife typically ranging from weeks to several months.

[0077] Third, because of the short half-lives of many radioisotopes,radioactively-labeled microspheres are typically usable only inexperiments of durations that are no more than a few weeks or months.

[0078] Fourth, commercially available automated gamma-ray countingequipment is NaI-based. Sodium-iodine (NaI) crystals provide a low-cost,sensitive gamma-ray counting system with intrinsically poor spacialresolution. As a result, researchers are limited in the number ofdifferent radioactive microspheres that can be accurately measured persample, due to overlap between the emission energies of availableradiolabels. Typically, researchers are limited to five radiolabels.Increasing the number of radiolabels measurements is done only at asignificant loss in sensitivity and specificity. (The measurement of theseparate radioactively-determined blood flows is performed bymathematically-based techniques. Namely, “matrix-inversion” analysis isperformed to remove the known “spill-over” between the emission spectraof various emitting species. A “cross-over” matrix is mathematicallysolved. These techniques are similar to the spectrographic analysis of apalette of dye-colored microspheres.) p Fifth, laboratory workers usingradioactively-labeled microspheres are exposed to radiation danger. Theradioactively-labeled microspheres are especially dangerous if theyenter into the human body by ingestion, respiration, or accidentalinjection. They are so small, and so numerous, so as to be incapable ofremoval. Accordingly, the costs, and risks, involved in minimizingradiation exposure can be substantial.

[0079] Sixth, licenses are required form various local, state, andnational Governmental regulatory agencies in order to transport,possess, use, and dispose of radioactive materials, includingradioactively-labeled microspheres.

[0080] Finally, and perhaps most critically, disposal of theexperimental animals poses significant problems, both logistically andfinancially. Because the entire animal carcass remains radioactive forsome time after use, and must be placed in a special low level radiationdump, to which dumps there is increasing public antipathy. The cost ofdisposal is becoming prohibitively high, recently ranging to as high as$750 U.S. or more per animal.

[0081] 2.4.4 The Earliest Measurements of Blood Flow With ColoredMicrospheres

[0082] Colored microspheres are primarily relevant to the presentinvention for showing (i) the increased flexibility in experimentalprocedures that may be realized when radioisotope-labeled microspheresneed not be timely extracted and measured, and (ii) the powerful ways bywhich, adequate time being had with no danger from radiation, themicrospheres can be chemically and even mechanically tailored on aparticular experimental protocol.

[0083] In 1967, polystyrene latex, radioactively-labeled microspheres(RM) were introduced for the measurement of regional perfusion. SeeRudolph AM, Heymann MA: The circulation of the fetus in utero: Methodsfor studying distribution of blood flow, cardiac output and organ bloodflow. Circ Res 1967;21:163-184.

[0084] One year later, Makowski, et al., introduced a blood withdrawaltechnique for the quantifying of regional blood flow. See Makowski E L,Meschia G, Droegemueller W. Battaglia F C: Measurement of umbilicalarterial blood flow to the sheep placenta and fetus in utero:Distribution to cotyledons and the intercotyledonary chorion. Circ Res1968;23:623-631.

[0085] In 1969, Domenech, et al., first validated the use of radioactivemicrospheres (RM) for the measurement of regional myocardial blood flow(RMBF). See Domenech R F, Hoffman J I E, Noble M I M, Saunders K B,Henson J R, Subijantos: Total and regional coronary blood flow measuredby radioactive microspheres in conscious and anesthetized dogs. Circ Res1969;25:581-596. Thereafter, this method has become the standardtechnique for the measurement of RMBF in various experimental settings.However, due to the precautionary measures needed to minimize radiationexposure, use of R M is restricted to specially licensed laboratories.As mentioned above, storage of the radioactive microspheres, as well asdisposal of radioactive waste, is expensive and presents a health andenvironmental hazard.

[0086] To avoid some of these limitations inherent to the R M method,U.S. Pat. No. 4,616,658 to Shell, et al. for NON-RADIOACTIVELY LABELEDMICROSPHERES AND USE OF SAME TO MEASURE BLOOD FLOW describes a methodfor measuring RMBF using non-radioactive, colored microspheres (CM).Later, Hale, et al., described a similar technique. See Hale S L, AlkerK J, Kloner R A: Evaluation of non-radioactive, colored microspheres formeasurement of regional myocardial blood flow in dogs. Circulation1988;78:428-434.

[0087] According to the techniques of Shell, et al., and of Hale, etal., microspheres may be (i) labeled with colored dyes, and (ii)subsequently visually identified and counted after recovery fromdigested tissue, either after separation therefrom or while stilltrapped in the tissue's capillaries. Shell, et al. also describelabeling microspheres by linkage to enzymes, particularly plant enzymes,and, after extraction from tissue, measuring the density ofenzyme-linked spheres by a measurement of colorometric density which isindicative of enzyme activity.

[0088] In the previous techniques using non-radioactively-labeleddye-colored microspheres (CM), tissue samples that have trapped, orcaptured, microspheres from the circulating blood of a live animal aresurgically harvested after euthanasia of the animal; and are thendigested by a combination of enzymatic and chemical methods. Aliquots ofthe microspheres trapped within a given sample are then counted in ahemocytometer by an investigator using light microscopy, or, in the caseof enzyme-linked microspheres, by measurement of colorometric density todetermine enzyme activity.

[0089] There are, however, significant limitations to these previouscounting techniques. First, RMBF is extrapolated from only a smallaliquot of the dye-colored microspheres (CM) actually trapped within thesample, thereby entailing a substantial statistical error in RMBFcalculations. Second, the use of a maximum of only three differentcolors (in the same experiment) has been validated in the literature,and then in only a small number of samples, whereas it is clearlydesirable to be able to make more than three measurements of RMBF inmany common experimental protocols. Third, there was considerablevariation in the diameter of the CM used in previous studies, asadmitted by Hale et al. Fourth, the prior methods require substantialtime for the tedious counting of individual dye-colored microspheres.Automation for optical counting is expensive, typically $40-50K U.S.circa 1993 . Fifth, in preliminary experiments, the inventors of thepresent invention found it almost impossible to distinguish visually thenine (9) commercially available microsphere colors in the reddishbackground of digested myocardium.

[0090] Recently, still another alternative non-radioactive method formeasuring RMBF was developed by Morita, et al. using X-ray fluorescenceexcitation of microspheres loaded with elements of high atomic number.See Morita Y, Payne B D, Aldea G S, McWattes C, Huseini W, Mori H.Hoffman J I E, Kaufmann L: Local blood flow measured by fluorescenceexcitation of non-radioactive microspheres. Am J Physiol1990;258:H1573-H1584. So far, only two different labels have beenreported to have been validated by comparison to radioactivemicrospheres (RM) after intracoronary injection in two dogs. The methodof Morita, et al. could be hampered by leaching of the label from themicrospheres over time. Another disadvantage is the need of asophisticated and extremely expensive equipment for X-ray excitation andfluorescence detection which is not commercially available.

[0091] The previous blood flow analysis methods employing dye-coloredmicrospheres, including the method of Morita, et al., require that thenumbers of microspheres per unit portion of a recovered tissue sampleshould be determined. Because the numbers of microspheres introducedwithin the blood [typically five to ten million (5-10×10⁶)], andcaptured within the capillaries of the tissue, are large in the countingtechniques, the actual numbers are commonly only estimated bystatistical sampling, which induces measurement error. Worse, even thedetermination of the numbers of dye-colored microspheres that are withinminute sub-samples is tedious and expensive, involving in the methods ofShell, et al., and of Hale, et al., manual or semi automatedobservations through a microscope.

[0092] In order to circumvent these limitations, it would be desirableif a new method of producing and/or using microspheres, and of measuringRMBF therewith, could support both (i) easy tissue processing (i.e.,digestion) and (ii) quantitative, automated, and easy counting of everymicrospheres within an individual sample. Such a new method woulddesirably be both economical and validated by a rigorous comparison toRM over a range of RMBF from 0 to 10 ml/min/g on many hundreds, orthousands, of individual myocardial samples. If such a method were to besuitably economical, reliable, easy to use, and devoid of significantdrawbacks, then it might find general use in the measurement andanalysis of diverse fluid flow and fluid mixing problems other than onlymedical problems.

[0093] 2.4.5 Blood Flow Measurement as Taught in the Certain PreviousPatents of Assignee Triton Technology, Inc.

[0094] The predecessor patents listed in section 2.3 above, assigned toTriton Technology, Inc. of San Diego, Calif., teach advanced dye-coloredmicrospheres, and the use thereof in blood flow measurement.

[0095] The methods of the related patents replace the “counting” of thenumbers of non-radioactively labeled microspheres during a use of suchmicrospheres in fluid flow analysis with, instead, a direct measurementof the amount of a colored, non-radioactive, dye that is carried by suchmicrospheres. The dye is removed from the recovered microspheres byelution or by simply dissolving the spheres. The solvent is thenanalyzed for dye content by absorbance or fluorescencespectrophotometry. Measurement of the amount of dye accomplisheddirectly by spectrographic methods is much easier and faster thancounting the numbers of microspheres, and accounts for all themicrospheres in the sample, and not just a small aliquot.

[0096] In particular, the (i) microspheres of the prior patents are dyedwith a color for which the quantitative photometric absorption oremission (fluorescence) spectrum is uniquely identifiable, (ii) thelabeled and dye-colored microspheres (CM) so created are introduced in afluid flowing into a volume serving as a reservoir of such fluid, (iii)after the introduction of the CM the concentration of dye within thefluid (concentration being the amount of dye per unit portion of fluid)is determined, (iv) the CM are recovered from a complete sample, not asmall aliquot of the sample volume into which the fluid containing theintroduced CM has flowed, (v) the colored dye is eluted or dissolvedfrom the recovered CM with a solvent, and (vi) the recovered dye isquantified by a spectrophotometric procedure. The relative amplitude ofthe photometric spectrum of the recovered dyes gives a quantitativeindication of the concentration of the dyes within the sample. Theconcentration of dye that was within the original fluid is normallysimilarly determined, i.e. by spectroscopy. The ratio of these twoconcentrations indicates the flow of the fluid within which the CM werepreviously resident into the volume relative to the overall flow offluid.

[0097] Fluorescent dye spectrum analysis offers some apparent advantagesfor CM applications, when compared to absorbance spectra analysis.Fluorescent dyes emit light isotopically and can be read off-axis (i.e.,at 90°) from the excitation source. Because reflection of the excitationlight is minimized at high angles, this serves to minimize noise in theform of extraneous light. The complete emission spectra can bede-convolved mathematically to analyze the areas under the waveforms ifthe increased sensitivity of “matrix inversion” peak analysis isrequired. Thus emission dyes may sometimes offer increased sensitivityover absorption dyes. However, statistical requirements for the minimumnumber of spheres required for a ‘significant’ measured value in a giventissue sample (typically 400 spheres per sample) partially offset themajor advantage in the sensitivity with which fluorescent, as opposed toabsorption, dyes may be detected. The primary advantage of fluorescentdyes appears to be their (i) excitation with a distinctive frequency ofradiation in order to fluoresce, and (ii) their potentially sharper, ornarrower, ‘peaks’, both making it theoretically possible the use widerpalettes of non-interfering colors than with purely absorption dyes.

[0098] When the (ii) introducing of the CM is into the circulating bloodof a live animal, and when the (iii) determining is of the concentrationof dye within the circulating blood, and when the (iv) recovering of theCM is from harvested animal tissue and blood by process of tissue andblood processing, then the (vi) measuring serves as a quantitativeindication of the concentration of the dye in the harvested tissue andblood, and thus of the flow of the blood within which the CM, and thedye, were contained to the harvested tissue.

[0099] The (i) coloring is typically of each of several different typesof microspheres: the microspheres of each type becoming labeled with anassociated one of a plurality of different colors. The quantitativephotometric spectrum of each color is both a) individually uniquelyidentifiable, and b) distinguishable from the photometric spectrum ofall other colors. When the (ii) introducing, and the (iv) recovering,are of the several different types of microspheres—either of which stepsmay transpire separated in time and/or space, and may be repeated—thenthe (vi) measuring in a spectrophotometric procedure is of the compositequantitative photometric spectra of the several recovered dyes.Accordingly, an expanded method includes the additional step of (viii)mathematically analyzing, or de-convolving, the composite spectra toaccount for spectral overlap between the individual spectra of theseveral dyes at each of several specific wavelengths, namely thewavelengths of the individual peak absorption (or emission) of theseveral dyes. The mathematical analysis preferably transpires by one ofseveral computerized mathematical processes, including matrix inversion.Each such individual spectrum is a quantitative indication of theconcentration of the dye associated with each individual type of labeledCM, and a corresponding indication of the flow of that (those) fluid(s)within which each type of CM was previously resident into the volume.

[0100] For emission spectrometry (as is taught in the predecessorpatents) the (vi) measuring and (viii) analyzing steps are both easy andsusceptible of automation. Despite their relative ease and simplicity,the steps are fully capable of accurately simultaneously determining theabsolute, and relative, abundances of a number of different types of dyewhich are within a corresponding number of different types of CM. Thesedifferent types of CM may be of different sizes, densities, shapes, orsurface characteristics—each of which may have correspondingly differentpropensities to lodge within tissue or other material (such as soil)contained within the volume. The different types of CM may have beenplaced within several different flowing fluids that were subsequentlymixed. The different types of CM may have been placed within the samestream of flowing fluid at different times. Accordingly, just oneautomated photometric analysis readily yields an abundance of temporaland spatial information regarding fluid(s) flow(s) and fluid mixing.Such abundant information is, in particular, eminently suitable formedical blood flow analysis including regional myocardial blood flow(RMBF) analysis, but is not so limited.

[0101] The methods of the predecessor patents were validated by itsproduction of quantitative results that are in close correlation to RMBFmeasured by 15 μm diameter radioactive microspheres after intracoronaryinjection in 4 pigs (r=0.98), and after intra-atrial injection in 4 dogs(r=0.97). The methods of the related predecessor patents are (i) fast,(ii) easy, (iii) susceptible of automation and (iv) cost-effective,while avoiding all problems related to radioactivity. However, tissuedigestion is still required.

[0102] When referring to microspheres, radioactive microspheres anddye-colored microspheres are called “types”. Each particularradioisotope, having a particular emission energies, that serves toradio-label microspheres of the radioactively-labeled type (i.e., RM) isspoken of as creating a “species” of that type. Similarly, eachdifferent color of dye-colored microspheres, or CM, is spoken of asbeing a particular “species” of CM.

[0103] The present invention will later be seen to improve upon the verynature multiple-species type of microspheres, and to contemplateautomatic accurate measuring of this new type of microspheres by neutronactivation analysis, and without the necessity of digesting anything, oreluting the marker label from the microspheres.

[0104] 2.4.6 The Desire and Need to Conduct Many Blood Flow TestsSimultaneously and/or in Series Sequence Before Subjecting a LaboratoryAnimal to Euthanasia and Harvesting Its Tissue

[0105] When an expensive laboratory animal is subjected to blood flowanalysis in order to evaluate the effects of various medical regimens,therapeutic and otherwise, there is a strong desire and need, based onefficiency of labor and reduction of cost, to accomplish as muchinvestigation at one time as is feasible. A first injection ofmicrospheres (of any type and species) typically later serves, when theanimal's tissue is harvested, as a bench mark of normal bloodcirculation to the organ of interest, and serves as a baseline orreference point. After this bench mark injection the experimentalprotocol begins. For example, a coronary artery perfusing a portion ofthe heart of an animal might be partially or completely clamped,simulating a coronary event (“heart attack”). Another, second, injectionof microspheres (of another type and/or species) generally serves toshow, when later detected in harvested tissue, a diminished (ornon-existent) blood flow, to the portion of the heart perfused by theclamped or partially clamped coronary artery. Other portions of theheart perfused by other coronary arteries will typically show no, oronly slight, changes in blood flow during the same intervention. Similarinjections at a number of subsequent times generally serves to define,when correlated with procedures and interventions performed on theanimal and/or the administration of drugs to the animal, exactly how theanimal's target organ is being perfused under several successive stepsof an experimental protocol conducted over a period of time.

[0106] In order to extract from the selected harvested tissue, and fromthe blood samples, the record of the various blood flow circulations atthe various times of the successive injections, it is necessary toseparately evaluate the presence (or absence) of each different type andspecies of microspheres as were injected into the animal upon successivetimes. With radioactive microspheres, some 7-8 overlapping types ofradioisotopes in common use each produce a distinct signature—permittingthereby an experimental protocol having up to 7-8 interventions and/ormeasurements at successive times. Of course, in accordance with the halflives of the radiolabels of the radioactive microspheres, theexperimental protocol, regardless of the number of steps, should alwaysbe terminated, and the radioactivity analyzed before the shortesthalf-life isotope becomes too weak to be useful.

[0107] Dye-labeled colored microspheres, or CM, are stable, and willsupport experimental protocols of long time duration. Presently, in themethods of the predecessor patents, typically more than five absorbance,and even more luminescent (fluorescent of phosphorescent), dye-coloredmicrospheres may be commonly reliably separately distinguished in thepresence of each other. The search goes on for palettes of even moredyes that are individually distinguishable from one another in theirspectrums of absorption or emission when a number of such dyes are allmixed together.

[0108] In general, a desirable characteristic sought for dyes used todye CM is a single very sharp peak absorbance or emission at awavelength suitably displaced from all other dyes of the pallet. Thespectrum of thousands of dyes have already been examined for thesecharacteristics, and the search continues. However, with existingspectrometer sensitivities, and with analytical software programs oftractable size and execution times, which, most importantly, produceaccurate quantitative results in the analysis of the individual spectraloutputs of several different dyes (derived from species of CM) mixedtogether, it has, to date simply not been possible, to identifycompatible dyes, and families of dyes, that number more thanapproximately one dozen. Accordingly, the number of separate steps inexperimental protocols using CM is currently, circa 1993, limited to adozen or less, and is more commonly and routinely (exotic dyes not beingused), limited to about seven.

[0109] Researchers would prefer that the (i) duration of theirexperiments, and (ii) the number of intervention steps, in anexperimental protocol should quite literally be unbounded. Although atotal lack of limits may not be possible, it would be useful if somescheme could be developed to permit the individual detection inharvested tissue of large numbers (more than 15-20) of species ofcolored microspheres—which CM are not subject to deteriorate over time.Such detections would obviously permit that effective, extensive,multi-step experimental protocols could ensue over indefinitely longperiods of time before it became necessary to perform euthanasia on theanimal, and harvest its tissue, to evaluate the effect on blood flow ofthe step-wise procedure. Accordingly, blood flow experiments ofconsiderable complexity, and many intervention steps, can be performedbefore the animal needs be subject to euthanasia, and its tissueharvested.

[0110] 2.4.7 Efficiency Issues When Conducting Great Numbers of BloodFlow Measurements By Methods as Are Taught in the Predecessor Patents

[0111] The flow measurement methods taught within the related patents—although a step forward over prior art radioactive microspheres andmeasurement methods—require certain steps that previousradioactivity-measuring methods using radioactive microspheres did notrequire. In previous radioactive methods, the abundance of radioactivemicrospheres (RM) in a unit sample of the harvested tissue is directlydeterminable by measurement of the level of gamma radiation emitted byeach species of RM present within the sample. This measurement isperformed without extracting the microspheres from the harvested tissuesample nor, for the matter, without extracting the radioactive substancefrom the RM.

[0112] When dye-colored microspheres (CM) are used instead, as is taughtwithin the related predecessor patents, then a necessary and ultimatestep is the quantitative assessment, by emission or absorbancespectroscopy, of the amount of dye that is within each species of thecollective CM that are within the harvested tissue sample. Thisparticular measurement step may be roughly as easily performed as areradioactivity measurements, and may be performed at roughly the same, orat lessor, cost than are radioactivity measurements.

[0113] However, in order to extract the dye(s) from the collectivemicrospheres that are within the harvested tissue sample, certainadditional process steps are required before the measurement step.First, the harvested tissue sample must be digested. Next, thedye-colored microspheres (CM) must be recovered from the digestate,normally by centrifugation and filtration. Finally, the dye(s) must beeluted or dissolved from the recovered CM.

[0114] Of these three additional steps, the second is by far the mostlabor intensive, and is therefore the most expensive. Both the digestionof the tissue, and the eluting of the dye from the recovered CM arechemical processes requiring only the addition of appropriate reagents.The recovery of the CM by centrifugation or filtration is, however, atime-consuming and attention-demanding process that must be carefullyperformed.

[0115] Accordingly, it would be useful if some improvement could be madeto the multi-step method of the related patents in order to (i) simplifyand/or (ii) automate the separation of the CM form the digestate, and/orthe dye form the separated CM, or else (iii) eliminate the requiremententirely. An improvement to CM would logically permit direct measurementof the CM that are within the harvested tissue sample in steps that wereeither (i) reduced in number, (ii) simplified, and/or (iii) automated,or semi-automated. Otherwise, a new type of microsphere is required, andthat is the subject of the present invention.

SUMMARY OF THE INVENTION

[0116] The present invention contemplates (i) carrying on or within acarrier, particularly microspheres, (ii) certain chemicals, and moreparticularly certain stable isotopes of elements, that are detectable byneutron activation analysis. The preferred microsphere carriers carrythe preferred stable isotopes into chemical admixtures or compounds, orinsert the isotopes into or upon objects or devices, where the isotopesthereafter serve as markers or tracers that serve to permanentlyidentify such admixtures, compounds, objects or devices.

[0117] This permanent identification continues after such change(s) andgross change(s) in form and/or in composition as may be occasioned bylapse of time, mechanical changes such as dissipation or wear ordeterioration, or chemical changes such as oxidation or reduction orexplosion. Basically, 1) the marking is as close to “forever” asanything yet known; and 2) the sensitivity of the tracing is as great asanything yet known, with the amount of tracers in the form of stableisotopes that are required for reliable detection being close to“infinitesimal”.

[0118] As a further aspect of the present invention, the preferredmicrosphere carriers may be tailored to their environment, such as bybeing, most commonly, biodegradable. The biodegradability is notprimarily so that the carrier microspheres may be removed from abiological environment at a time after they have served to deliver thepreferred stable isotope(s) into that environment. Indeed, themicrospheres will typically do no harm even should they persist forever:they will typically have no effect on the biological organism and theywill never interfere with any subsequent detection and/or measurement ofthe isotopes by neutron activation analysis. Instead, the microspherecarriers are—in a manner totally unlike prior biological usages ofmicrospheres as tracers where the microspheres were required to remainintact—preferably involved in, or even consumed by, some biologicalprocess(es) regarding which the condition(s), rate(s), and/or site(s) orsuch process(es) are of interest. An experimenter simply monitors thelocations, and distributions, of the stable isotopes as an indication ofwhat is happening to the microspheres. As a simple example, the carriermicrospheres might be made of, or bound together by, some protein orsugar. When carried into the gut of an animal, these microspheres willpersist intact for varying periods depending upon conditions.Observation and detection of the locations, and the time distribution,of stable isotopes within the gut of the animal—either asmicrosphere-sized agglomerations or as dissipated throughout some volumeof the animal's gut—clearly indicates what is happening to the proteinor sugar microspheres within the animal's gut.

[0119] The concept of observing (by sensitive neutron activationanalysis) the concentration and distribution of marker isotopes borne onmicrospheres where the microspheres themselves are not invariant, butare instead tailored to and/or interactive with their environment, isapplicable to more than biological organisms. For example, microspherescarrying upon their surfaces a marine anti-fouling chemical agent, aswell as (within or upon the surface of the microspheres) certain markerisotopes, may be placed into a marine hull paint, imparting anti-algalor other properties to the paint. Later analysis of samples from theenvironment in which a ship painted with such marine hull plate isdocked will detect the marker isotopes with an extreme sensitivity thatis much, much greater than any ability to directly detect theanti-fouling chemical. To such extent as the marker isotopes come to befound in the environment then the microspheres, and the anti-foulingchemical agent may also assumed to be in the environment.

[0120] Thus a principle concept of the present invention is that acarrier, preferably microspheres, should carry a marker or tracer thatis detectable by neutron activation analysis, preferably one or morestable isotopes, into a target and, occasionally, beyond.

[0121] 1. The Carriage of Marker Elements Upon or Within Microspheres

[0122] The carriage of the chemicals, preferably certain stable isotopesof elements, upon the carriers, preferably microspheres, is important tothe permanence of the marking.

[0123] In accordance with one of its aspects, the present inventioncontemplates separating (i) chemical markers for, and the chemicalmarking of, targets from (ii) the mechanical and/or chemical means bywhich the chemical markers are emplaced and retained on or within thetargets. To repeat, the number of different admixtures, compounds,objects or devices in the world that may, as targets, desirably bemarked, and particularly selectively marked, is vast. Meanwhile, thenumber of chemicals that may usefully serve as markers—and particularlycertain stable isotopes of certain elements optimum for detection byneutron activation analysis in accordance with a separate aspect of theinvention—is limited. It is hard, even impossible, to directlychemically or mechanically associate the limited number of markerchemicals (the stable isotopes of elements) with each of the vast numberof different targets, thereby directly “marking” the targets. Inaccordance with the present invention, any requirement to chemically ormechanically “match” the (limited) markers to the (many) targets isobviated by the use of marker carriers: to wit, microspheres.

[0124] Not only do the microspheres carry (mechanically, or chemically,or both chemically and mechanically) the preferred marker(s), thecarrier microspheres may readily be tailored (chemically, ormechanically, or both chemically and mechanically) to the intendedapplications environment. Once the “system” of any particularapplication is set in place, there is no need to ever separate 1) themicrospheres from the marked target, nor 2) the marker chemical(s) (thestable isotopes of certain elements) from the microspheres. Indeed, thisseparation may be effectively impossible.

[0125] When and if the marked target or, more typically a minusculeportion or remnant thereof, ever comes to be assayed by neutronactivation analysis which serves to detect the marker chemical(s) (thestable isotopes) that the target originally contained or attached (bydint of the incorporation or affixation of microspheres which themselvescontain or affix the marker chemicals), then this assay, or analysis,will be performed on combined material of (i) the target, (ii) themicrospheres and (iii) the chemical markers (the isotopes) in common,and all together.

[0126] This analysis of everything taken together and in gross presentsno problem. The material of the target, and also the base material ofthe microspheres before association of the marker chemical(s), will notnormally contain any appreciable amount of the marker chemicals, whichmarker chemicals are preferably stable isotopes of elements rare innature. Accordingly, substantially only those marker chemicals (thosestable isotopes) that were originally added (via their microspherecarriers) to the target will be detected by neutron activation analysis.

[0127] This detection will transpire with exceedingly great sensitivity,meaning that a target may be indelibly permanently marked by but minuteamounts of chemical markers (i.e., minute amounts of one or more stableisotopes of suitably rare elements, or at least elements that areuncommon within the target).

[0128] 2. Markers Detectable by Neutron Activation Analysis

[0129] In accordance with another of its aspects, the present inventioncontemplates markers that are strongly reliably detectable, particularlyin the presence of and commingled with biological material, by processof neutron activation analysis.

[0130] In accordance with the present invention, these (i) preferredchemical markers that are strongly (i) detectable, and (ii) reliablyuniquely identifiable, in biological samples by process of neutronactivation analysis consist essentially of: stable isotopes of selectedelements. With greater specificity, the preferred chemical markers meetthe following criteria:

[0131] 1) They are stable isotopes of certain elements selected inaccordance with further criteria, next following.

[0132] 2) For any given target and target sample, the proposed stableisotope(s) (if not also the element(s)) should be rare, and thereforeall detection counts following neutron activation analysis can beattributed to presence of the marker element isotope(s).

[0133] 3) Following neutron activation, the theoretical specificactivity(ies) “is” of the marker-tracer-isotope(s) should be at least1×10¹⁰ disintegrations per minute per kilogram ofmarker-tracer-isotope(s). (This term “specific activity”, or “s”, willbe seen to appear within the neutron activation equation later discussedin the DESCRIPTION OF THE PREFERRED EMBODIMENT section of thisspecification.)

[0134] Some of the factors that can contribute to this specific activitywill be seen to be (i) a high natural fractional abundance (f), and (ii)a high neutron cross-section (σ≈100 barns), as both of these termsappear in the neutron activation equation later discussed in theDESCRIPTION OF THE PREFERRED EMBODIMENT section of this specification.

[0135] Although not absolutely necessary, following neutron activation,the daughter nuclide of the ideal marker-tracer-isotope will preferablyemit only one primary photon having an energy greater than 100 keV witha yield of 100%.

[0136] Moreover, and again although not absolutely necessary, thehalf-life of the daughter nuclide (t_(½)) should be (i) at least twodays, and more preferably greater than four days, but (ii) no longerthan two years, and preferably no longer than one month; thereby (i)allowing shorter-lived background activity to decay before counting thesample, while at the same time (ii) not creating long-lived radioactivewaste due to the marker-tracer itself.

[0137] There are a limited number of isotopes of elements in theperiodic table that meet all these criteria, and all these preferences.Gold is one example. However, many isotopes—particularly those of thelanthanide family—partially meet most of these criteria and preferences,and can easily achieve the required specific activity. For example, anisotope with a low natural fractional abundance (f=1%) can still achievethe required specific activity following neutron activation if itpossesses a higher neutron cross-section (σ≈1000) or if the isotope isenriched to achieve a fractional abundance of 1000% Overall, there areat least 25 isotopes of elements suitable for use as markers-tracers.

[0138] The use of stable isotopes of elements as marker-tracersessentially means that the marker-tracers are, short of atomictransmutation, indestructible. The elemental marker-tracers are foreverdetectable, and re-detectable—should an appropriately intense neutronflux, an appropriately prolonged decay counting period, and anappropriately sensitive radioactive decay event sensor be employed—atconcentrations that are as small or smaller than those detectable by anyother known analysis process. The sensitivity is thus down to thenaturally occurring level of the marker-tracer in the sample.

[0139] Herein lies the relationship between the major aspects of thepresent invention. In accordance with the present invention, these (i)quite marvelous indestructible superbly-detectableunambiguously-identifiable marker-tracer elements are (ii) carriedmechanically within (and more rarely, chemically upon) microspheres. Themicrospheres may be of selectively predetermined size(s), andselectively predetermined chemical composition and/or surface coating(ex of the marker-tracer isotopes), as is important in some biologicalexperiments. The microspheres may be (further) chemically or (stillfurther) mechanically combined with chemicals or other compounds thatfixate the target of interest. For example, it is well known that thesurfaces of microspheres may be coated with antigens to promote theselective uptake of the microspheres into certain tissues in biologicalexperiments.

[0140] Accordingly, the present invention involves a method of(typically permanently) marking targets with stable isotopes of elementssuitable for neutron activation analysis by act of carrying these stableisotopes within microspheres that are chemically and/or physicallyadopted to the targets.

[0141] 3. Marking for Neutron Activation Analysis

[0142] In greater detail, the marking of the present invention, which isnormally permanent, is applied to diverse admixtures and/or compoundsand/or devices. The marking is with chemical markers that are detectableby neutron activation analysis.

[0143] The present invention broadly satisfies the need for identifyingobjects or admixtures or compounds or the like for forensic purposes.“Marking” in accordance with the present invention transpires by thesimple expedient of adding chemical markers in the form of one or moresubstances that are strongly detectable by neutron activation analysisto objects or to admixtures or to compounds, thus providing the objectsor admixtures or compounds with a permanent “signature”. The marking isnormally at very low levels, and is inconsequential to the function(s)of the objects or admixtures or compounds. Once marked, the chemicalmarkers cannot be (i) reasonably eliminated nor (ii) masked fromdetection by neutron activation analysis.

[0144] The chemical markers are preferably non-radioactive, stable,isotopes of selected elements. In accordance with the present invention,the admixtures or compounds or devices or the like are not markeddirectly with the stable isotopes but are instead marked by anintermediary carrier or carriers each bearing one or more stableisotopes. The marked intermediary carriers are, in particular andpreferably: microspheres. The stable-isotope-marked microspheres are inturn used to mark (or to re-mark) something else, for example abiological sample.

[0145] In accordance with the fact that the detectable isotopes arestable, the marking and re-marking may transpire over time, in multiplesteps, and/or at howsoever many indirect steps as are desired. Forexample, a single microsphere appropriately marked with plural stableisotopes, or, alternatively, a selected palette of singly-markedmicrospheres, as do encode for, by way of example, a unique identity (asmay represent any of time, location, source, owner, intended deployment,etc. etc.) may be combined with, by way of example, an adhesive andapplied to, by way of example, a security fence. In this example, animalfur or human clothing that is later determined by sampling and by(neutron activation) analysis to bear a pattern of traces of theselected isotopes may be assumed to have come into contact with thefence wheresoever, and at a later time then, the fence was marked.

[0146] As another example, batches of explosives may be individuallyuniquely marked directly by (i) microspheres containing multipleisotopes, and/or by (ii) selected combinations of single-markedmicrospheres. In this example, explosive residue that is later sampledand analyzed to show a pattern of some specific isotopes may be assumedto have originated in a batch of explosive so marked with theseparticular isotopes.

[0147] Isotope-marked microspheres are useful in biologicalinvestigations including, for example, flow measurement such as, inparticular, the measurement of (i) regional organ blood flow and (ii)airway flow. Furthermore, isotope-marked microspheres are useful in drugdelivery studies including (i) particulate distribution in drugdelivery, (ii) immunoassays, and (iii) high-throughout screening fordrug discovery.

[0148] In one of its preferred embodiments as isotope-markedmicrospheres, the present invention presages a new generation ofhigh-precision alternatives to traditional, radio-labeled life scienceproducts. The microspheres labeled with stable (non-radioactive)isotopes are used in an analogous fashion to their radioactivecounterparts. However, the analysis of the tracer isotope(s) in samplesof interest is performed by use of neutron activation technology. Thebiological marking system so based is highly sensitive, accurate andeasy to use, providing in many cases information with improvedsensitivity and specificity well beyond that achievable by colorometricmarkers and traditional radiolabels.

[0149] 4. Chemicals (Stable Isotopes of Elements) Suitably Subjected toNeutron Activation Analysis as Makers, and the Detection of MarkerChemicals (Stable Isotopes of Elements) by Neutron Activation Analysis

[0150] The preferred chemical markers of the present invention aretypically permanent and without appreciable susceptibility to changewithin, or removal from, the compounds, objects or devices in which theybecome embedded, to which they become attached, or with which theybecome associated. If desired—and even though microspheres may melt and,indeed, the marker isotopes themselves may turn into liquids or gases atsufficiently high temperature—it can usually be arranged that neitherthe microspheres nor the marker isotopes can be removed without causingsuch a change to the sample—such as melting—as is thereafter permanentlyunambiguously recognizable. Note that it is possible to, in certaincases, intentionally liberate the microspheres. For example, suppose therelative contributions of (1) gasoline and (2) alcohol as are bothpresent in gasohol to particulate contamination of the atmosphere mightbe assessed if each of the (1) gasoline and (2) alcohol components wasseparately marked in accordance with the present invention.

[0151] If necessary the chemical marker, typically a stable isotope ofan element, may be associated with a succession of one or more “markercarriers”, and “carriers of marker carriers” which are most commonly inthe physical form of microspheres, and layered microspheres. Themicrospheres, and microsphere layers, may have chemical propertiestargeted on the environment of use such as, by way of example, to(harmlessly, and at low numbers) chemically compound with the target,making subsequent removal while preserving the integrity of the targetall but impossible.

[0152] Conversely, it should be understood that, where unauthorizedremoval of the markers is not an issue, as in biological experiments,then the marker (i.e., one or stable isotopes in or on a microspherecarrier) may intentionally be made inert, and non-reactive with theintended environment of use. The (typically microsphere-based) “markercarriers”, and (the typically layered microsphere) “carriers of markercarriers”, may be considered to be a “bridge” between the markers (thestable isotopes) and the intended environment of use.

[0153] Detection of the marker chemicals may transpire, andre-transpire, at any time, and from time to time, and both before andafter the occurrence of events, by subjecting and re-subjecting thechemicals, and any matrix in which they are then present or commingled,to neutron activation analysis. The neutron activation analysis istypically non-destructive to the compound, object or device within whichthe marker chemical is placed. Consider, by way of comparison, thatneutron activation analysis has been deemed safe for use on all thediverse materials as may be contained in luggage carried on airlines.

[0154] In accordance with the well-known principles and method ofneutron activation analysis, the marker chemicals (and surroundingsubstances) are bombarded with neutrons so that atoms of these chemicalsbecome excited to a higher energy level. The subsequent radioactivedecay of these marker chemicals from their excited atomic statestranspires over time as discrete events that may readily bedetected—essentially individually detected—by nuclear scintillationcounters and like devices. The marker chemicals are accordingly detectedessentially at the levels of individual atoms, and over a vast range ofdensities. Depending upon the radioactive half lives of the excitedmarker chemicals, and the amount of marker chemicals present in thesample being analyzed, analysis may typically take some hours or days oreven longer. And analysis requires, of course, an intense neutronsource, such as is normally associated with a research reactor. However,by the well-understood quantum mechanical principles of radioactivedecay, quantitative measurement of the number of marker chemicalmolecules or atoms present may essentially be obtained, with sufficienttime, to any desired degree of mathematical accuracy.

[0155] Notably, the marker chemicals may be detected by neutronactivation analysis over a vast range of densities. A neutron is aneutral particle. Therefore, neutron flux can effectively penetratedense material. The resultant radioactive emission of the tracer is inthe form of a photon. A photon is an energetic, massless particle thatcan be emitted from dense material of a reasonably sized sample (i.e.,some few grams or tens of grams) with little self-attenuation. As aresult, this technology can provide a detection method requiring minimalsample preparation and that can be used in a wide variety ofapplications.

[0156] The preferred marker chemicals are not common, let alone incombination. The marking thus has a superb “signal-to-noise”: once anobject, admixture or compound is marked then it can be, depending uponthe density of the marking, detected indefinitely long at anindefinitely great dispersion.

[0157] Most typically in biological and in other applications of thematerials and methods of the present invention there is at any one timebut one potential “target” in the “field of view”, and the only interestof the investigator is in (unambiguously) identifying this target and/orits abundance the sample. However, it should be understood that thepresent invention works to detect both (i) multiple targets, and (ii)strong targets in the presence of weak targets, occurring concurrently.

[0158] For example, consider that multiple marked targets should be,sometimes over a period of years, “loosed” upon the environment.Although such “marking” of multiple targets in the environment as maycumulatively occur over years and decades may be assumed to be innocuousas regards any environmental impact, some thought may usefully beapplied as to just how to mark these multiple targets. For example, sometens of thousands of batches of explosives might be used coextensivelyin a single rock quarry over a period of decades. In the matter of howto uniquely encode, or mark, each of these batches both informationtheory, and industry standards and assigned usage, assume a greater rolethan it is common for, by way of comparison, bar codes. It is possiblefor a well-thought-out scheme to, by use of 25+ tracer-marker isotopes,uniquely identify each of many millions of targets each of which isdifferently marked, sometimes even while in the presence of othertargets. In the example of the stone quarry, residues of theexplosive(s) most recently used would be expected to be most pronouncedin the newly shattered stone, with most stone that had been stronglymarked by explosive blasts some years earlier having since been hauledaway. It thus becomes possible in some situations, and by judiciousplanning, to detect individual targets even if other targets areconcurrently present to a greatly reduced degree. All residualtracer-markers un-associated with the dominant target clearly constitute“noise” to the detection, and unique identification, of the dominanttarget.

[0159] The marker chemicals of the present invention are not destroyednor permanently changed by being subjected to neutron activationanalysis, and can be analyzed and re-analyzed. Neutron activationanalysis is completely non-destructive of the marker chemicals analyzed.This characteristic supports that samples of marked compounds anddevices preserved at, usually, time of manufacture may be analyzed fortheir detail salient characteristics at any time in the future, and thatunknown samples may likewise be analyzed and re-analyzed as may proveimportant, for example, in verifying and re-verifying their detailnature and their value as evidence in a judicial proceeding.

[0160] For example—and continuing with the example of explosivesalthough any of oil, pollutants and art are equally valid—a minutesample of an original, marked, item or compound of, for example, aparticular batch of an explosive may be preserved. If the explosiveresidue of a bomb site later analyzes to the particular chemical markersor series thereof that this heretofore untested sample is supposed tocontain, then it is a simple and straightforward matter to test andre-test both reference and sample until all possible information isderived from both.

[0161] 5. The Packaging and Use of Marker Chemicals Detectable byNeutron Activation Analysis, Including in Microspheres and Including forUse in Blood Flow Analysis

[0162] The present invention contemplates that chemicals detectable byneutron activation analysis may be incorporated within microspheres, andthat families of such chemicals may be used to lend unique identities tomultiple microspheres, and families of microspheres.

[0163] As well as their obvious physical properties of size, weight,density, etc., the microspheres may have other chemical propertiesincluding, inter alia, a surface incorporation of antibodies orproteins. The microspheres so marked become lodged by the circulatingblood within selected tissues of an animal during blood flow analysisexperimentation. They may remain there, substantially imperviousness toalteration, indefinitely. If and when tissue containing the markedmicrospheres is ever harvested, it may be subjected to neutronactivation analysis, normally in bulk and without any preparationwhatsoever, so as to determine the absolute and relative abundances ofthe microspheres, and each different type thereof, by measurement of theradioactive decay, which is at a different energy signature for eachtype of chemical marker as is associated with a corresponding type ofmicrosphere.

[0164] Notably, the sample not only need not be purified or eluted orotherwise treated, but is substantially impervious to contamination, andneed not even be isolated (save only from new marker chemicals). Thesample may commonly be put in a closed non-contaminated vial or othercontainer, and the entire vial subjected to neutron activation analysis.Containing no marker chemicals itself, the container vial has noinfluence on the measurement results.

[0165] Quick, easy and accurate measurement of blood flow to diversetissues as is performed in blood flow analysis experimentation is thusobtained: marked microspheres inserted into the bloodstream of a liveexperimental animal are subsequently detectable in the harvested tissuesof the animal by to such quantitative accuracies as are a indicative ofthe former flow of blood containing the microspheres to the tissues ofthe animal.

[0166] These any other aspects and attributes of the present inventionwill become increasingly clear upon reference to the following drawingsand accompanying specification.

BRIEF DESCRIPTION OF THE DRAWINGS

[0167]FIG. 1 is a prior art diagram of the principles of neutronactivation analysis.

[0168]FIG. 2 is an emission spectra from a 5 g liver segment containingsix labeled microspheres (samarium, rhenium, iridium, gold, antimony,and lanthanum); the spectra showing the presence of the tungsten monitorwhich is added to the sample-vial to account for potential variations inthe neutron flux and sodium which is the major contributor to backgroundnoise for most biological samples.

[0169]FIG. 3 is a Table 1 providing the important physicalcharacteristics of eight different isotopes that are suitable as labelsfor microspheres to measure regional perfusion.

DESCRIPTION OF THE PREFERRED EMBODIMENT

[0170] 1. Non-Radioactive, Stable, Isotope-Labeled Microspheres

[0171] 1.1 General Sequence of Making and Using Isotope-LabeledMicrospheres

[0172] In one of its aspects the present invention is expressed innon-radioactive, stable, isotope-labeled microspheres. Preferably someeight or more different, stable, isotopically-labeled microspheres areto be made available to experimenters.

[0173] It is intended that isotope-labeled microspheres shall typicallycome in 2 ml glass serum bottles containing approximately 5 millionspheres. The microspheres are 15 μm in diameter and are suspended innormal saline containing 0.05% Tween 80 and 0.01% Thimerosal as abacteriostat.

[0174] The tissue sample vials in which the tissue samples are to beplaced are contaminant-free. The preferred sample vials are made frompolypropylene, cleaned to remove trace contamination and calibrated foruse in the particular activation and counting system of the assayist,which quite reasonably requires the use of its own microspheres andvials must be used for the assay.

[0175] The assayist typically charges a per-sample assay fee for theassay service. The results of the assay are normally reported as thetotal disintegration per minute (dpm) measured in each sample for eachcorresponding microsphere label. If preferred assayist BioPhysics AssayLaboratory, Inc., 280 Wellesley Avenue, Wellesley Hills, Mass. 02481(Phone/Fax: (781) 239-0501) [“BioPAL”] is used, then the absolute bloodflow (ml/min/g) measured in each sample can also be calculated for anadditional assay fee.

[0176] 1.2 Metallic Isotope Dye Preparation

[0177] Stable isotope labeled microspheres can be fabricated in a numberof ways, but the first step is to make a solvent-soluble metalliccomplex of the desired stable isotope. Typically this can be done byfirst preparing a metallic AC-AC (acetylacetonate) solution using amethod similar to that of Brown, et al., J. Organic Nuclear Chemistry,Vol. 13, pp. 119-124, 1960.

[0178] For example, Lanthanum AC-AC complex can made by dissolving 1.0gm of a metallic salt lanthanum (LaCl₃), in 40 ml of de-ionized waterusing 0.1 normal hydrochloric acid (HCL) to effect. Next, blend thesolution using a stirring plate and a stir bar while adjusting the pH to5.0 using 0.2 normal sodium hydroxide. When the mixture is thoroughlymixed, stir in 10 ml of 2,4 pentanedione and allow the mixture to fullyhomogenize (approximately 10 minutes). After the mixture is thoroughlyhomogenized, further neutralize the solution using sodium hydroxideuntil a stable pH of 6.5-7.0 is obtained.

[0179] The solution is then placed in a constant temperature bath at65°-70° C., with no further agitation for one hour. After one hour, coolthe solution down to room temperature (requiring approximately 1.5hours).

[0180] Add 40 ml of benzene to the mixture and thoroughly homogenize bystirring for ten minutes. Transfer this mixture to a separating funneland allow to stand until the metallic complex has floated to the top.Pour-off the liquid-phase and set aside for further processing. Transferthe metallic complex to a dish to air dry under a stream of 65° C. air.Add 20 ml of benzene to the recovered liquid phase from the separatingfunnel step. Recover any remaining metallic complex by again using theseparating funnel and this material to the drying dish. Dispose of theliquid-phase after the second separation step. Store the dried metallicAC-AC material in a covered container at 0° C.

[0181] 1.3 Fabricating Microspheres

[0182] Making of stable-isotope labeled polystyrene latex microspherescan be accomplished in one of several ways. Microspheres labeled with astable-isotope metallic complex can be fabricated using a method similarto that described in “Neutron-Activated Holium-166-Poly (L-Lactic Acid)Microspheres: A Potential Agent for the Internal Radiation Therapy ofHepatic Tumors”, J. Nuclear Medicine, R. J. Mumper, U. Y. Ryo, and M.Jay , Vol. 20 No. 11, 2139-2143, 1991.

[0183] Alternatively, commercially manufactured polystyrene latexmicrospheres can be ‘dyed’ using a solvent-solution of thestable-isotope metallic complex as described in the publication “UniformLatex Particles”, Seradyn Inc, Indianapolis Ind., 46206, author Dr.Leigh Bangs. Microspheres suitable for dyeing are available from BangsLaboratories, Fishers Ind.

[0184] In addition, stable isotope labeled microspheres can befabricated by covalent or non-covalent coating of the outside surface ofthe microspheres with the metallic complex in a manner similar to theNEN-Trac® radioactive microspheres manufactured by New England Nuclear,Boston Mass. 02118-2512. (NEN-Trac® is a trademark of New EnglandNuclear.)

[0185] For example, lanthanum labeled polystyrene microspheres can befabricated by first making a 3% solution of polyvinyl alcohol (PVA).This is done by adding 2.4 g of polyvinyl alcohol to 800 ml ofde-ionized-water in a 1-liter beaker. Place the beaker on a stir plateand put a stir bar in the beaker. Cover the top of the beaker withplastic wrap to prevent evaporation and set the stirring to a fairlyrapid rotation rate. Allow the mixture to stir until thoroughlyemulsified which will take overnight. Keep the mixture covered and atroom temperature.

[0186] The microspheres are fabricated by adding the components togetherwith constant stirring under a nitrogen gas environment. This can beaccomplished by fabricating a mixing-box that can be sealed and purgedwith nitrogen gas. The mixing box has provisions for an externalstirring system to stir the vinyl solution continuously while themicrospheres are forming. The box is continuously purged with a low flowof nitrogen during the first hour of processing.

[0187] Dissolve 2 g of polystyrene material in 30 ml of chloroform in a50 ml glass beaker. It will take about one hour for the polystyrene todissolve, vortexing or agitation will speed the process.

[0188] Measure out 0.5 g of the previously fabricated metallic AC-ACcomplex and place in a small beaker inside the mixing box. Pour 300 mlof the PVA solution into a 500 ml glass beaker, then place the beaker inthe mixing box. Thirdly, place the beaker of chloroform/vinyl in themixing chamber. Seal the mixing box and purge the box with nitrogen.Begin stirring the PVA solution in the 500 ml beaker at 1000 rpm. Usinga glass syringe and a tube through the mixing-box side, transfer themetallic complex (lanthanum AC-AC, in this example) to thechloroform/vinyl solution and allow the mixture to fully dissolve.Slowly add the metallic complex plus chloroform/vinyl solution to thestirring PVA (1000 rpm). Allow this mixture to stir for a minimum of onehour under the nitrogen atmosphere, and afterwards continue stirring foran additional 12 hours in air inside a fume hood. This additional timeis to insure that all the chloroform has evaporated away.

[0189] The microspheres are next washed and sorted by size. Washing isaccomplished by re-suspending the microspheres in a 1-liter beakercontaining solution of de-ionized water with 0.05% Tween 80 as asurfactant. Place the beaker on a stir plate, place a stir bar in thebeaker and stir the mixture at a medium rate. Place the tip of anultrasonic homogenizer (“sonicator”) in the beaker and sonicate themicrosphere mixture at low power for 5 minutes while stirring. Transferthe microsphere mixture to multiple 50 ml centrifuge tubes andcentrifuge the tubes for 10 minutes at 1500 g (2500 rpm on mostbench-top centrifuges).

[0190] Aspirate the supernate down to a safe level above themicrospheres and re-suspend with 30 ml of 0.1 normal hydrochloric acid(HCL) to remove any unincorporated metallic complex from the outsidesurface of the microspheres. Sonicate each of the 50 ml tubes for 30seconds and then fill each tube to the top with the HCL solution.Centrifuge the tubes again for 10 minutes at 1500 rpm, aspirate the acidsolution. Re-suspend the microspheres in water plus 0.05% Tween 80,vortex mix, centrifuge, aspirate down to the microsphere pellet. Repeatthe last step three times to remove any remaining acid. After the lastwash step do not aspirate but instead combine the microspheres from allthe tubes into a clean 1-liter glass beaker. The microspheres can now besized using a mechanical microsiever (Gilson Company,Inc, Worthington OHModel SS-5 or equivalent). For the measurement of blood flow in mostspecies, microspheres of 10 to 15 μm in diameter have been found to beoptimal.

[0191] The sized microspheres are washed in alcohol, centrifuged and thesupernate aspirated away, after which they are allowed to air-dryovernight. The dried microspheres are then re-suspended in an injectablesaline solution containing 0.05% Tween 80 at a concentration of 2.5million microspheres per milliliter. The Tween 80 is a surfactant thatis used to minimize aggregation of the microspheres in solution. If themicrospheres are going to be stored for later use, 0.01% Thimerosalshould be added to the solution as a bacteriostat.

[0192] 1.4 Characteristic Uses of Isotope-labeled Microspheres SoProduced

[0193] Stable isotope-labeled microspheres so produced have a widevariety of potential applications, most notably to the life sciencecommunity is its use in regional blood perfusion and particle depositionstudies. Normally at least eight different isotopic labels, at aminimum, can be simultaneously assayed by neutron activation.Investigators use the stable isotope-labeled microspheres in ananalogous fashion to their radioactive and dye-elution-based(colorometric) counterparts, the major difference being that samples ofinterest are subsequently sent to BioPAL for analysis of their tracer(s)content, with the results of the assay typically being returned to thecustomer within a week. The standard report sheet of BioPAL provide thecustomer with the number of disintegrations per minute (dpm) measuredfor each labeled set of microspheres, which is analogous to thetraditional radioactive method. Given additional information provided bythe customer, BioPAL can also calculate the blood flow (ml/min/g) foreach tissue sample.

[0194] The stable isotope-labeled microsphere product and servicetypically offers a cost-savings of from 50-200% depending on the methodbeing compared, improved sensitivity (with one microsphere resolution),re-assay and archiving ability, and the elimination of the risk ofsample contamination and lose associated with other nonradioactivemethods.

[0195] 2. Details of the Assay of the Isotope-Containing Microspheres

[0196] 2.1 Mathematical Basis of Neutron Activation Analysis

[0197] Assays in accordance with the present invention are based onneutron activation analysis, typically as performed by a serviceprovider (i.e., someone other than the experimenter and user of thelabeled microspheres). The preferred assayist/analyst—saidBioPAL—employs customized neutron activation protocols for themeasurement of trace and ultra-trace elements.

[0198] Neutron activation analysis to measure the concentration ofstable isotope-labeled microspheres is a two step process. The firststep requires exposing the sample to a high neutron field sufficient togenerate a strong radiation signal from each isotope label.

[0199] The general principle underlying neutron activation is that anincident neutron is captured by an atom forming a radioactive daughternucleus. The number of radioactive emissions is directly proportional tothe mass of the parent isotope. See the diagram of FIG. 1.

[0200] In practice, a sample is exposed to a flux of neutrons, φ, for agiven time, t. The specific activity, s, induced in any parent nuclidecan be calculated from the formula:

s=6.02×10²⁶ φσfA ⁻¹(0.5)^(t) ^(₁) ^(/t) ^(_(½)) [1−(0.5)^(t/t) ^(_(½)) ]

[0201] where: s=specific activity in disintegrations per unit mass (s⁻¹kg⁻¹), φ=flux of neutrons (m⁻²s⁻¹), σ=cross-section for neutroninteraction with the parent nuclide (in barns, or m²), f=fractionalabundance of the parent nuclide, A=atomic weight of the parent element,t¹=time between activation and counting (hours), t=activation period(hours), and t^(½)=half-life of daughter nuclide (hours).

[0202] In accordance with the present invention, a marker will be markedwith an isotope that following neutron activation will generate atheoretical specific activity (s) of which exceeds 1×10¹⁰disintegrations per minute per kilogram of tracer, as appears in theabove equation for specific activity, s, induced in any parent nuclideduring neutron activation.

[0203] 2.2 Qualities of Preferred Isotopes, and the Preferred Isotopesof the Present Invention for Use as Markers Suitably Assayed by NeutronActivation Analysis

[0204] The neutron activation process of the present invention willpreferably activate nearly all individual members of all species ofisotopes present in the sample. Therefore, an ideal stable isotope foruse as a tracer in biological and other studies would have the followingcharacteristics (as previously discussed in the SUMMARY OF THE INVENTIONsection of this specification) in order to be most effectively measuredagainst background noise:

[0205] 1) The marker element has a stable isotope.

[0206] 2) For any given sample, the proposed stable isotope should berare, and therefore all detection counts during neutron activationanalysis can be attributed to presence of the tracer.

[0207] 3) The fractional abundance of the isotope (f) should be 100%.(See the term “fractional abundance” in the neutron activation equationof section 2.1, above.)

[0208] 4) The isotope should have a high neutron cross-section (σ>100barns) (“barns” have the unit of m²). (See the term “σ” in the neutronactivation equation of section 2.1, above.)

[0209] 5) Following neutron activation, the daughter nuclide should emitonly one photon having an energy greater than 100 keV with a yield of100%.

[0210] 6) Because of their unique energy signatures upon decay from anexcited state, the daughter nuclide must be, and innately will be,individually distinguishable from one another.

[0211] 7) The half-life of the daughter nuclide (t_(½)) should be (i) atleast two days, and more preferably greater than 4 days, but (ii) nolonger than 1 year, and more preferably no longer than 1 month; thereby(i) allowing shorter-lived background activity to decay before countingthe sample, while at the same time (ii) not creating long-livedradioactive waste due to the tracer itself.

[0212] Also as previously discussed in the SUMMARY OF THE INVENTIONsection of this specification, only Gold (Au) substantially meets allthese criteria. Other tracer elements must be selected in accordancewith their ability to substantially meet, to a greater or a lessorextent, most criteria.

[0213] In choosing an appropriate tracer, consider that, for biologicalsamples, sodium, potassium and chloride provide the greatestcontribution to the background signal given their short activatedhalf-lives (²⁴Na t_(½)=15 h, ⁴²K t_(½)=12 h, ³⁸Cl t_(½)37 m). Comparedto an ideal tracer, this background activity is low following thethree-day decay period without having a significant loss in signal fromthe activated tracer. Moreover, following a five week decay period, mostbiological samples plus the ideal tracer content would be covered underNuclear Regulatory Commission regulations for exempt concentrations (10C.F.R. §32.11), and therefore can be returned to the researcherincluding a researcher at a non-NRC licensed laboratory.

[0214] There are a number of isotopes, particularly isotopes in thelanthanide family, that largely meet the characteristics of an idealtracer.

[0215] One such isotope is gold, having a fractional abundance of 100%(¹⁹⁷Au) and a cross-section of 98.7 barns. The daughter nuclide of gold(¹⁹⁸Au) emits seven photons, one of which has an energy of 411 keV and ayield of 95.5%, while the other six photons all yield below 2%. Thehalf-life of ¹⁹⁸Au is 2.69 days.

[0216] Other usable isotopes include, but are not limited to, stableisotopes of antimony, lanthanum, samarium, europium, terbium, holmium,ytterbium, lutetium, hafnium, tantalum, tungsten, rhenium, osmium,iridium, scandium, bromide and still others.

[0217] 2.3 Performance of Neutron Activation Analysis on SamplesContaining Isotope-Labeled Microspheres in Accordance With the PresentInvention

[0218] Accordingly, an ideal isotope for use as a microsphere label willform a radioactive nucleus that would be short-lived and emit agamma-ray during the decay process. The energy of the gamma-ray isdiscrete and distinct for each stable isotope. In general, samples willrequire an exposure of 1.0×10²⁰ neutron m⁻² to generate a useful signal.As a result, mid-size research reactors that can generate a neutron fluxin the range of 10¹⁷ to 10¹⁹ neutrons m⁻² s⁻¹ and will provide the bestsource of neutrons. Other sources, such as sealed tube generators andaccelerators, cannot currently achieve the necessary neutron flux orrequire too much energy and are therefore not cost effective. Researchreactors have the added advantage of being able to generate a wideneutron flux beam suitable for activating large numbers of samplessimultaneously.

[0219] Several methods can be used to introduce samples to the neutronfield, such as robotic belts and pneumatic delivery systems. Forexample, a well-designed pneumatic delivery system can activate onaverage 4,000 samples per day. Most mid-size research reactor would beable to accommodate as many as 10 systems operating independently,thereby providing a daily theoretical throughput of 40,000 samples.

[0220] The second step is effectively measuring theemissions-of-interest following neutron activation. In addition to thestable isotope labels, the activation process will also activate otherisotopes present in the sample adding background noise to the signal,thereby potentially decreasing the sensitivity of the assay. Given theirrelative high concentration in biological samples, sodium and chloridepose the greatest concern. However, given their short activatedhalf-lives (²⁴Na t_(½)=15 h, ³⁸Cl t_(½)=37 m) compared to themicrosphere labels (t_(½)=2 to 100 d), this background activity will below following a 2-3 day decay period.

[0221] It is also important to note that desalting methods exist and canbe adapted to help remove NaCl and other contaminates fromsamples-of-interest. However, in most cases this step is not needed.Following neutron activation, samples are then stored in a secured,NRC-licensed, area for the decay of short-lived background activity.

[0222] Specialized, high-resolution detection equipment can than be usedto identify and measure emitted gamma-rays. The number of emittedgamma-rays is directly proportional to the total mass of the parentisotope, and therefore is proportional to the total concentration of thelabeled microspheres contained in the sample. The sensitivity andspecificity of a radiation detection system is dependant on many factorsincluding background interference, intrinsic efficiency of the detector,source-detector geometry, sample size, and the times for irradiation,decay, and counting. The complexity of adjusting these variables isintensified when trying to optimize the system for multipleelements-of-interest simultaneously. Because quantitative gamma-rayspectrometry of low energy photons can be complicated by scatteredelectrons, resulting from higher energy photons generating backgroundcounts due to Compton scatter. Specifically, a photon entering adetector has a high probability of interacting with an electron. Afraction of the photon's energy is given to the electron, while thephoton recoils with the remaining energy. This phenomena is calledCompton scatter. Short ranged Compton electrons will deposit theirenergy within the detector and are represented as counts. These countscan confound the quantitative assessment of a complex gamma spectrum,such as those generated following neutron activation of a biologicalsample. Several methods can be used to reduce Compton contributions. Onesuch method uses a Compton suppression system consisting of a largecrystal ring detector surrounding a primary, high resolution detector.As the recoiled photon exits the primary detector, it has an opportunityto deposit its energy with the second ring detector. Deposition of theCompton electron within the primary detector should occur simultaneouslywith the absorption of the recoiled photon within the ring detector.Operating the two detectors in anti-coincidence can reduce the Comptoncounts by a factor of 10, while the loss of counts in the total energypeak will reduced by less than 0.05%. Given recent improvement isdetector technology and computer electrons, such a system can bedesigned and automated to process up to 1,000 to 2,000 samples per day.Having several systems working in series, such a counting system caneasily handle the output of activated samples.

[0223] So functioning, the aforementioned BioPAL, in particular, offersthe biomedical research and industrial communities access to the powerof neutron activation technology for the measurement of trace elements.Using their isotope-labeled microspheres of their own distribution(manufactured by Triton Technology, Inc. in San Diego, Calif., inaccordance with the one of the method taught above), the energy of thegamma-ray is discrete and distinct for each stable isotope. Specialized,high-resolution detection equipment is then used to identify and measurethe emitted gamma-ray. The number of emitted gamma-rays is directlyproportional to the total mass of the parent isotope.

[0224] The sensitivity of any nuclear reaction can be estimated usingEquation 1. For Samarium, the potential reaction is ¹⁵²Sm(n,γ)¹⁵³Sm;t^(½)=1.93 days. Therefore, using a flux of 8.0×10¹⁶ (m⁻²s⁻¹) and anirradiation time of 10 minutes, followed by a decay time of 2 days, aspecific activity of 2.1×10¹² disintegrations per second per kilogram isobtained for ¹⁵³Sm.

[0225] Therefore, this technique BioPAL (or anyone following theteaching of this specification) can determine less than 10 ⁻¹² kg (1 ng)of Samarium using standard counting equipment. Given recent improvementsin detector technology and computer electronics, this sensitivity can beat minimum improved by an order-of-magnitude.

[0226] 2.4 Summary of the Intertwining of Neutron Activation Analysisand the Present Invention, With Particular Emphasis on Support forBiological Assays

[0227] Neutron activation analysis is well known for its excellentsensitivity and elemental specificity for the simultaneous measurementof trace elements. See Anal Chim Acta 165, 1 (1984); Anal Chem 63, 1143(1991); and Anal Chem 65, 1506 (1993).

[0228] Unlike other methods, such as atomic absorptionspectrophotometry, neutron activation is not chemically or physicallydestructive. Therefore, samples can be re-assayed or undergo otherchemical analyses following neutron activation. Neutrons are highlypenetrating, therefore this method can be used to assay samples having awide range of material compositions and densities. In addition, thisassay can be structured so that it is completely self-contained, therebyminimizing the possibility of sample contamination and sample loss.Despite these advantages, this technology is not widely used because oflimited availability of neutron sources, traditionally high activationcosts, and long experimental turn-around times.

[0229] A major focus on applications of the present invention has thusbeen on the development of a new generation of high-precisionalternatives to traditional, radioactive life science products. Thepreferred marked microspheres labeled with stable isotopes are used inan analogous fashion to their radioactive counterparts. The assay of thestable tracer(s) in samples of interest is performed by an analystagent—an assayist—that is located at a source of neutron flux (normallya research reactor), and that performs the assay using neutronactivation technology for a fee. This method of local experimentationcoupled with a remote, service-bureau-provided assay, has been found tobe both accurate and easy to use.

[0230] In many cases, this approach provides information with improvedsensitivity well beyond that achievable by colorometric markers andtraditional radiolabels. Given the economies of scale resultant from thelarge number of samples that the assayist analyzes daily, the assay maybe competitively priced with what it would cost an investigator to dohis or her own analysis based on an alternative, prior art, markingsystem. The assay can usually be customized, supporting where relevantcomputations based in the quantitative measurement of trace elements invarious material compositions. For example, when the assayistunderstands that it he assaying a harvested tissue for a sequence of A,B, C, D marked microspheres as are indicative of blood flow to thetissue at times T₁, T₂, T₃, T₄ in a blood flow analysis experiment, thenhe may well report the results as (relative) blood flow, as well as thequalitative levels of abundance.

[0231] 3. Advantages of the Present Invention

[0232] The core technology of the present invention is neutronactivation analysis. Neutron activation analysis is well known for itsexcellent sensitivity and elemental specificity for the simultaneousmeasurement of trace elements. Stable-labeled research products in theform of chemicals suitable to neutron activation analysis, with anaccompanying vender neutron activation analysis service, offers the lifescience community the following advantages:

[0233] The use of radioactive and hazardous reagents may be totallyavoided. The assay of the present invention is non-radioactive for theresearcher while requiring none of the hazardous reagents associatedwith colorometric measurements. Therefore, researchers and biotechnologycompanies do not require an Nuclear Regulatory Commission (or equivalentforeign regulatory authority) license nor will they need to purchase andmaintain expensive in-house laboratory equipment.

[0234] The present invention accords even the most modest researchactivity full access to a highly sensitive and specific assaytechnology. Namely, neutron activation provides a highly sensitivemeasurement of the stable-labeled products in accordance with thepresent invention. Unlike other methods, assay in accordance with thepresent invention does not require extensive sample preparations thatcan result in sample contamination or sample loss. In many cases, (i)the stable-labeled products subsequently subjected to (ii) an assayservice, both in accordance with the present invention, offers increasedsensitivity well beyond that achievable by colorometric markers andtraditional radiolabels.

[0235] The present invention accords multiple labels. Nature hasprovided a reasonably large number of stable isotopes that canpotentially be used—some more effectively than others—to label researchproducts and can be efficiently and simultaneously assayed by neutronactivation.

[0236] The present invention offers flexibility to researchers whendeveloping experimental protocols. In addition to easily permittingmultiple labels, neutron activation is not chemically or physicallydestructive. Therefore, samples can undergo other chemical analysesfollowing the assay.

[0237] The present invention permits that the original sample should bearchived, and re-assayed if, as and when desired. Assayed samples can bestored for future reference and re-assayed, if necessary, at a laterdate. Furthermore, samples can be re-assayed to provide additionalsignal amplification retrieving information that would otherwise be lostusing alternative technology.

[0238] The present invention accords reduced experimental start-upcosts. Researchers and research institutions do not have to learn a newtechnology, nor invest in expensive hardware, nor purchase high-pricedreagents, nor secure additional laboratory labor for the performance ofassays in accordance with the present invention.

[0239] The present invention readily permits the evaluation of highlyconcentrated or dense samples. Neutrons are highly penetrating.Therefore, highly concentrated samples or samples of varying materialcompositions such as bone or teeth can readily be assayed. This assay,therefore, eliminates signal quenching errors associated with bothbeta-radiation counting (i.e. 3H, 14C, 32P, 35S) and opticalmeasurements.

[0240] 4. Non-Radioactive Stable Isotope-Labeled Microspheres ForPhysiological Studies

[0241] Non-radioactive, stable, isotope-labeled microspheres inaccordance with the present invention provide the biological researchcommunity with a cost-effective alternative to the use of radioactive,and or colorometric (dyed) microspheres for the measurement of regionalorgan blood flow and particle deposition studies.

[0242] The experimental method for using stable-labeled microspheres isidentical to the traditional radioactive method. See Prog Cardiovasc Dis20, 55 (1977); and Basic Res Cardiol 80, 417 (1985). The method of thepresent invention differs only in the assay of the microspheres isperformed at the location of a neutron source, normally a researchreactor, normally by a provider of neutron activation analysis assayservices such as said BioPhysics Assay Laboratory, Inc., 280 WellesleyAvenue, Wellesley Hills, Mass. 02481 (Phone/Fax: (781) 239-0501)[“BioPAL”] [the “assayist”].

[0243] At the close of the experiment, tissue samples of interest arecollected, weighed and sealed in tracer-free polypropylene sample vials.If blood flow measurements are to calculated (ml/min/g), then bloodsamples are sent to the assayist for analysis, who uses neutronactivation technology for the measurement of microsphere content. Theresearcher can typically expect the results of the assay within sevenworking days via e-mail.

[0244] The assay procedure of the assayist is not chemically orphysically destructive and the measurement can be repeated andre-repeated on the samples or any of them at a later time. See, forexample, Anal Chim Acta 165, 1 (1984). Therefore, samples may bereturned to the researcher for other analysis or archived for futurereference. The ability to re-assay stored samples is a major advantagefor pharmaceutical companies in meeting FDA/GLP requirements.

[0245] 5. Non-radioactive Immunochemical Assay Kits

[0246] The present invention is embodied in non-radioactiveimmunochemical assay kits supportive of the non-radioactive methods ofthe present invention in immunochemical applications. In this case themicrospheres are coated or impregnated with biological substances, forexample monoclonal antibodies, as well as the stable isotopes that serveas tracers/markers.

[0247] The inventors of the present invention are actively developing,circa 1999, new products adapting neutron activation technology tomeasure stable, isotopically labeled monoclonal antibodies (r otherbiologically active compounds) for use in immunochemical applications.This technology will allow for the simultaneous evaluation ofmultiple-labeled monoclonal antibodies (greater than two) in a singlesample and should prove to be more cost effective then currentfluorescent activated cell sorting technology. Neutron activation is notchemically or physically destructive and the measurement can berepeated. See Anal Chim Acta 165, 1 (1984). Therefore, samples may bereturned to the researcher for other analysis or archived for futurereference. This ability to re-assay the same sample will provide a majoradvantage for pharmaceutical development. Preliminary findings suggestthat this technology will also provide improved sensitivity well beyondthat achievable by current fluorescent markers and the traditionalradioimmunoassays.

[0248] 6. High-Throughput Screening for Drug Discovery

[0249] The present invention supports and permits customizedhigh-throughput screening for drug discovery and evaluation.

[0250] Neutron activation analysis provides the researcher withcapabilities not readily available with other assay technologies. Forexample, neutron activation analysis can sensitively and accuratelymeasure several (ultimately ten or more) stable isotope labels,simultaneously. This offers the potential of being able to measuremultiple assays in a single well. Screens designed to look forfunctional relationships among related molecules are also possiblewithin a single well.

[0251] Because neutron activation is nondestructive to the sample, smallpeaks found in the first pass can be reactivated for longer times toincrease assay sensitivity. This variable sensitivity feature of neutronactivation may be able to detect hits that might be missed by othertechnologies.

[0252] After the initial measurement, samples can be archived for longperiods of time. Since the labels are atomic elements they will notbreak down. If it becomes important to re-assay a sample(s) at a laterdate (months to years later), this is possible.

[0253] Neutrons can penetrate solid tissues, so multi-label screening ofintact tissue derived from in vivo models is possible.

[0254] The above features of neutron activation can potentially speed upthroughput by a factor of at least ten, lower costs, allow for “smarter”assay designs, do direct in vivo assays and save time.

[0255] 7. Microspheres Tailored to, and Interactive With, theEnvironment Into Which They Transport the Marker Isotopes

[0256] Microspheres in accordance with the present invention are mostcommonly inert, and stable, in the environment into which theytransports and deliver the marker isotopes. They may be, however, madetailored to, and intentionally interactive with, this. This interactionis normally over a longer time, or in the face of later-arisingconditions, than necessary to transport the marker isotopes into theenvironment of interest.

[0257] The tailored microsphere carriers are most commonlybiodegradable. This biodegradability is not directed to eliminating themicrospheres from a biological environment at a time after they haveserved to deliver the preferred stable isotope(s) into that environment.Instead, the reaction of the microsphere carrier with the biologicalenvironment—so as to degrade, or whatever—is important as an indicationof the time(s) and location(s) of biological reactions not between theenvironment and what the microspheres carry, but between the environmentand what the microspheres themselves are. This is totally unlike theprior biological usages of microspheres as tracers where themicrospheres were required to remain intact. The tailored microspheresare involved in, or even consumed by, some biological process(es). Thereason that this is of interest is that the condition(s), rate(s),and/or site(s) or these biological process(es) may be determined bymonitoring over time the locations, and distributions, of the stableisotopes as an indication of what is happening to the microspheres.

[0258] As a simple example, the carrier microspheres may be made ofsugar, much like a confectioner's hard shell candy coating, or of aprotein, for example casein (from milk). When carried into the gut of ananimal, these microspheres will persist intact for varying periodsdepending upon conditions. Observation and detection of the locations,and the time distribution, of stable isotopes within the gut of theanimal—either as microsphere-isotopes sized agglomerations or asdissipated throughout some volume of the animal's gut—clearly indicateswhat is happening to the protein or sugar microspheres within theanimal's gut. For example, the digestion of sugar microspheres maydiffer in animals (including people) having stomachs that are relativelymore or less acidic. And casein is involved, or course, in lactoseintolerance which manifest as the total or partial inability of somepeople to digest this protein.

[0259] This concept is broad: microspheres may be made to be tailored toand/or interactive with their environment for any legitimate reason. Forexample, microspheres can carry specific materials regarding whichdissemination and distribution of which in the environment, sometimesover a period of years, is desired to be studied, and known. The samemicrospheres will, in accordance with the present invention, also carrymarker isotopes. The microspheres need not, and most often do not,bio-degrade. However, as they move in the environment so also do thecarried isotopes move. These isotopes can be detected by neutronactivation analysis with much, much greater sensitivity, and accuracy,than the specific materials. Where the microspheres are so also are thespecific materials. Accordingly, detection and abundance measurement ofthe microspheres is equivalent to the detection and abundancemeasurement of the specified materials.

[0260] 8. Use of the Nonradioactive, Stable-Isotope-Labeled,Microspheres of the Present Invention in Blood Flow Analysis

[0261] As the above sections make clear, non-radioactive,stable-isotope-labeled, microspheres in accordance with the presentinvention have many uses. The microspheres are, however, of exemplaryusefulness in blood flow analysis, and, accordingly, their use in thisapplication is explained in detail.

[0262] 8.1 Introduction to the Use of Microspheres for Blood FlowDetermination

[0263] Radioactively labeled polystyrene microspheres have been used tomeasure regional blood flow since the technique was first described inthe late 1960's. See Circ Res 21, 163 (1967); Circ Res 23, 623 (1968);Circ Res 25, 581 (1969); and J Appl Physiol 31, 598 (1971). A review ofthe technique has been published by Heymann and co-workers. See ProgCardiovasc Disease 20, 55 (1977)

[0264] Recently, nonradioactive colored have been used. See Circulation78, 428 (1988); and Circulation 83, 874 (1991). Additionally,fluorescent microspheres have been used in these experiments. See Am JPathol 103, 292 (1981); Kidney Intl 20, 230 (1981); Am J Physiol 251,H863 (1986); J Autonomic Nerv Syst 30, 159 (1990); and Am J Physiol 262,H68 (1992).

[0265] Previous techniques have called for the recovery of not only themicrospheres, but also the absorbance/fluorescent dyes that themicrospheres contain, from harvested tissue samples of animals, allowingblood flow quantitation to be performed using instrumentation such asspectro-fluorometers or fluorescence spectophotometers.

[0266] For example blood flow measurements using the Dye-Trak™microspheres product of Triton Technology, Inc., San Diego have beenvalidated in side-by-side comparisons with radioactively labeledmicrospheres (Dye-Trak™ is a trademark of Triton Technology, Inc.). Seethe aforementioned Kowalik, et al. “Measurement of Regional MyocardialBlood Flow With Multiple Colored Microspheres”, Circulation, Vol. 83,No. 3 March, 1991. The two techniques exhibited equivalent maximumdetection sensitivity and the correlation between flow measurements wasexcellent. The Dye-Trak™ microspheres are presently available in morethan five colors, allowing effects of multiple physiological variablesto be studied in the course of a single experiment.

[0267] The present invention has been seen to negate the steps ofdigesting the sample, recovering the microspheres, and eluting theabsorbance or fluorescence dye from the recovered microspheres.Nonetheless that no dye is extracted from (recovered)stable-isotope-labeled microspheres in accordance with the presentinvention, the same (i) detection sensitivity and (ii) accuratecorrelation between flow measurements as with colorometric microspheresis enjoyed. This is because the present invention teaches a way toaccurately quantitatively measure the amount of the marker element thatis within the microspheres (and thus, indirectly, the abundance of themicrospheres themselves) while this element is still (effectivelypermanently) within the microspheres.

[0268] Note that not only that the number, but the rate, of decay countscan be, and commonly is, measured.

[0269] 8.2 Stable-isotope-labeled Microspheres

[0270] The microspheres of the present invention, as are theirpredecessor products, are uniform polystyrene microspheres with nominaldiameters of 10 μm or 15 μm. They are commonly supplied as suspensionsin 10 mL of 0.15 M NaCl with 0.05% Tweens^(SM) 80 and 0.01% thimerosal.These 0.2% suspensions contain either 3.6×10⁶ microspheres (10 μmdiameter) per mL or 2.5×10⁶ microspheres (15 μm diameter) per mL. Themicrospheres or each reagent contain a single marker element having astable isotope. The decay energy(ies) of the excited form(s) of thiselement is (are) well resolved from corresponding decay energies of theexcited form(s) of all of the other marker elements. The microsphereshave a relative density of 1.05 g/mL, which is close to that of redblood cells (1.10 g/mL).

[0271] 8.3 Uniformity of Stable-isotope-labeled Microspheres

[0272] The size uniformity of the microspheres determined by a suitableanalytical method for determining particle size distribution. Each lotcomplies with the following specifications: Microsphere DiametersStandard Deviation  9.5-10.5 μm <.45 micron 15.0-16.0 μm <.45 micron

[0273] The actual measured diameter for each lot of microspheres iscommonly printed on the product label.

[0274] 8.4 Stability of Stable-isotope-labeled Microspheres

[0275] The stability of the stable-isotope-labeled microspheres isexcellent in both dry and aqueous environments. Only a powerful organicsolvent, or destruction of the microspheres, will suffice to separateout the marker isotope(s).

[0276] The stability in aqueous suspension has been evaluated for thefollowing adverse conditions: 1) leaching of the isotopes into theaqueous medium during storage, and 2) reproducability of the signalobtained from the microspheres after prolonged storage. Each lotcomplies with the following specifications: 1) no loss of isotopes frommicrospheres after six months storage in aqueous medium; and 2) nochange in stable-isotope-labeled signal during neutron activationanalysis after six months storage in aqueous medium.

[0277] 8.5 Color Coding Corresponding to the Stable Isotope With Whichthe Microspheres Are Marked

[0278] Because microspheres marked with different stable isotopes arenot thereby generally rendered visually distinguishable from another,the different stable-isotope-marked are preferably different dyed,thereafter supporting human visual recognition of, and distinctionbetween, the differing species of marked microspheres.

[0279] In accordance with these and other possible variations andadaptations of the present invention, the scope of the invention shouldbe determined in accordance with the following claims, only, and notsolely in accordance with that embodiment within which the invention hasbeen taught.

What is claimed is:
 1. A microsphere marked with a non-radioactivestable isotope of an element which isotope and element can be renderedradioactive for detection by neutron activation analysis.
 2. The markedmicrosphere according to claim 1 wherein the microsphere physicallyholds the element; wherein the marking is by physical associationbetween the element and the microsphere.
 3. The marked microsphereaccording to claim 1 wherein the microsphere chemically binds theelement; wherein the marking is by chemical association between theelement and the microsphere.
 4. The marked microsphere according toclaim 1 marked with a plurality of non-radioactive stable isotopes. 5.The marked microsphere according to claim 1 deployed within a samplewherein the microsphere is marked with an isotope that is so rare withinmaterial of the sample that essentially all detection counts duringneutron activation analysis can be attributed to presence of theisotope, and essentially none result from the material of the sample. 6.The marker microsphere according to claim 1 marked with an isotope thatfollowing neutron activation will generate a theoretical specificactivity (s) of which exceeds 1×10¹⁰ disintegrations per minute perkilogram of tracer, as appears in the equation for specific activity, s,induced in any parent nuclide during neutron activation: s=6.02×10²⁶φσfA ⁻¹(0.5)^(t) ^(₁) ^(/t) ^(_(½)) [1−(0.5)^(t/t) ^(_(½])) where:s=specific activity in disintegrations per unit mass (s⁻¹ kg⁻¹), σ=fluxof neutrons in m⁻²s⁻¹, σ=cross-section for neutron interaction withparent nuclide (m²), f=fractional abundance of the parent nuclide,A=atomic weight of the parent element, t₁=time between activation andcounting (hours), t×activation period (hours), t_(½)=half-life ofdaughter nuclide (hours).
 7. The marked microsphere according to claim 1marked with a plurality of isotopes that, following neutron activationanalysis, are by their unique energy signatures upon decay from anexcited state individually distinguishable from one another.
 8. Themarked microsphere according to claim 1 marked with an isotope that,following neutron activation analysis, produces a daughter nuclidehaving a half life (t_(½)) greater than one day.
 9. The markedmicrosphere according to claim 1 marked with an isotope that, followingneutron activation analysis, produces a daughter nuclide having a halflife (t_(½)) less than two years.
 10. The marked microsphere accordingto claim 1 marked with an isotope that, following neutron activationanalysis, produces a daughter nuclide having a half life (t_(½)) of atleast two days and shorter than one month.
 11. The marked microsphereaccording to claim 1 marked with an element from the group consisting ofgold, antimony, lanthanum, samarium, europium, terbium, holmium,ytterbium, lutetium, hafnium, tantalum, tungsten, rhenium, osmium,iridium, scandium and bromide.
 12. The marked microsphere according toclaim 1 color coded in accordance with the element with which it ismarked.
 13. The marked microsphere according to claim 1 used in bloodflow analysis, the marked microsphere characterized in that it may bedetected within a tissue sample resultant from blood flow analysis. 14.The marked microsphere according to claim 1 used in high-throughputscreening for drug discovery and evaluation, the marked microspherecharacterized in that it may be detected within a sample suitable forsaid high-throughput screening for drug discovery and evaluation. 15.The marked microsphere according to claim 1 comprising: a biodegradablemicrosphere body; with which is associated the non-radioactive stableisotope of an element only during the perpetuation of the physicalmicrosphere body, the isotope he being loosed when the body biodegrades.16. The marked microsphere according to claim 15 wherein thebiodegradable microsphere body consists essentially of a substancedigestible in the gut of a higher animal.
 17. A microsphere marked witha non-radioactive stable isotope of an element the presence of whichisotope which can be detected by neutron activation analysis, theisotope following neutron activation generating a theoretical specificactivity (s) of which exceeds 1×10¹⁰ disintegrations per minute perkilogram of tracer, as appears in the equation for specific activity, s,induced in any parent nuclide during neutron activation: s=6.02×10²⁶φσfA ⁻¹(0.5)^(t) ^(₁) ^(/t) ^(_(½)) [1−(0.5)^(t/t) ^(_(½])) where:s=specific activity in disintegrations per unit mass (s⁻¹ kg⁻¹), φ=fluxof neutrons in m⁻²s⁻¹, σ=cross-section for neutron interaction withparent nuclide (m²), f=fractional abundance of the parent nuclide,A=atomic weight of the parent element, t₁=time between activation andcounting (hours), t=activation period (hours), t_(½)=half-life ofdaughter nuclide (hours).
 18. A method of preparing a material forsubsequent identification comprising: labeling a material with amicrosphere marked with an non-radioactive stable element the presenceof which isotope, and element, can be detected by neutron activationanalysis; wherein neutron activation analysis of the marked material ora portion thereof will serve to identify the isotope and element, andthus the marked microsphere, and thus the marked material.
 19. Themethod according to claim 18 wherein the marking of the microsphere isby physical association of the material and the microsphere.
 20. Themethod according to claim 18 wherein the marking of the microsphere isby chemical association of the material and the microsphere.
 21. Themethod according to claim 18 wherein the material labeled is anexplosive.
 22. The method according to claim 18 wherein the material islabeled with a microsphere marked with a non-radioactive stable isotopeof an element from the group consisting of gold, antimony, lanthanum,samarium, europium, terbium, holmium, ytterbium, lutetium, hafnium,tantalum, tungsten, rhenium, osmium, iridium, scandium and bromide. 23.A material labeled by the method according to claim
 18. 24. A method ofmeasuring blood flow to tissue by use of microspheres collectivelymarked with a non-radioactive stable isotope that may be induced toemission by stimulation with a neutron flux, the method comprising:introducing a great multiplicity of non-radioactivestable-isotope-marked microspheres within the circulating blood of ananimal; harvesting a tissue sample of the animal which, due to previousretention of microspheres from the circulating blood, contains amultiplicity of marked microspheres therein; subjecting the harvestedtissue containing the multiplicity of microspheres to a source ofneutron flux in a process of neutron activation analysis so as tothereby induce effectively all, each and every one, of the stableisotopes within the multiplicity of marked microspheres to assume anelevated energy state from which each isotope will eventually decay;counting decays per unit time of stable isotopes from their elevatedstates as an indication of the presence of the stable isotopes, and thusof the marked microspheres containing the isotopes, the abundance ofisotopes and marked microspheres serving as an indication of theproportion of the multiplicity of marked microspheres that were withinthe harvested tissue sample relative to the great multiplicity of markedmicrospheres that were introduced into the circulating blood of theanimal; wherein the indicated proportion is also an indication of theblood flow of the animal to the tissue at the time of the introducing,and before the time of the harvesting.
 25. The method of measuring bloodflow to tissue according to claim 24 wherein the marked microspheres areof different types each containing isotopes that may be individuallydistinguished from other isotope types during neutron activationanalysis; and wherein the detecting and measuring of the magnitude ofthe isotope decays is an indication of not only the proportion, but alsothe absolute number, of the multiplicity of marked microspheres thatwere within the harvested tissue sample relative to the number of thegreat multiplicity of marked microspheres that were introduced into thecirculating blood of the animal.
 26. A method of (i) labeling samplescoupled with (ii) a subsequent identification by process of neutronactivation analysis of at least one of the samples so labeled, thesample labeling and identifying method comprising: uniquely marking eachof a plurality of types of microspheres with at least an associated oneof a plurality of non-radiative stable isotopes each of an element thepresence of which can be detected by neutron activation analysis;uniquely labeling each of a multiplicity of samples with at least anassociated one type of a plurality of types of microspheres; irradiatingan entire sample of unknown identity, which sample is uniquely labeledwith at least an associated one type of the plurality of types ofmicrospheres, with radiation sufficient to excite all the stableisotopes of all the types of microspheres to an associated higher energystate; and detecting decay events of the at least one excited stableisotope within the irradiated sample so as to uniquely identify themicrosphere type with the sable isotope is associated, and from thisidentified microsphere type, the uniquely associated sample; wherein asignature of radioactive decay by each stable isotope from itsassociated excited state is uniquely detectable from among decays of allother isotopes from their excited states; wherein, because (i) thesignature of each stable isotope is uniquely detectable and (ii) eachtype of microsphere is uniquely marked with at least one stable isotope,so also is each type of the plurality of types of microspheres uniquelydetectable from among all other types of microspheres; wherein, because(i) each type of the plurality of types of microspheres is uniquelydetectable from among all other types of microspheres, and (ii) eachsample is uniquely labeled with at least an associated one of aplurality of types of microspheres, so also is each sample uniquelydetectable from among all others of the multiplicity of samples.
 27. Thesample labeling and identifying method according to claim 26 wherein theunique marking of each of a plurality of types of microspheres is with apredetermined quantity of the least an associated one of a plurality ofnon-radiative stable isotopes; wherein the unique labeling of each of amultiplicity of samples is with a predetermined quantity of at least anassociated one type of a plurality of types of microspheres; and whereinthe detecting of the decay events of the at least one excited stableisotope within the irradiated sample so as to uniquely identify themicrosphere type with the stable isotope is associated furthercomprises: detecting the rate of the decay events as an indication ofthe abundance of the isotopes, which is in turn indicative of theabundance of the microspheres, within the sample.